CN104804094A

CN104804094A - CA6 antigen-specificity cytotoxicity conjugate and application method thereof

Info

Publication number: CN104804094A
Application number: CN201510114668.3A
Authority: CN
Inventors: G·佩恩; P·宗; D·塔瓦雷斯
Original assignee: Immunogen Inc
Current assignee: Immunogen Inc
Priority date: 2005-08-22
Filing date: 2005-08-22
Publication date: 2015-07-29

Abstract

All embodiments of the invention disclose a cytotoxicity conjugate containing a cell binding agent and a cytotoxic agent, treatment composition containing the conjugate, a method for inhibiting cell growth and treating diseases by the conjugate as well as a kit containing the cytotoxicity conjugate. Particularly, the cell binding agent is a monoclonal antibody or epitope-binding fragments of the monoclonal antibody and recognizes and binds CA6 glycotope. The invention further relates to a DS6 with humanized or resurfaced versions, an anti-CA6 mouse monoclonal antibody and epitope-binding fragments of the anti-CA6 mouse monoclonal antibody.

Description

CA6 antigen-specific cytotoxic conjugate and application method thereof

To be the applying date be the application on 08 22nd, 2005 and denomination of invention is the divisional application of the 200580051368.X application for a patent for invention of " CA6 antigen-specific cytotoxic conjugate and application method thereof ".

Technical field

The present invention relates to mouse-anti-CA6 sugar epi-position (glycotope) monoclonal antibody and humanization or resurfaced version (humanized or resurfaced versions).The present invention also relates to the epitope binding fragments (epitope-binding fragments) of anti-CA6 sugar epi-position monoclonal antibody, and the humanization of anti-CA6 sugar epi-position monoclonal antibody or the epitope binding fragments of resurfaced version.

The invention further relates to the cytotoxic conjugate comprising Cell binding agent and cytotoxic agent, comprise the therapeutic composition of described conjugate, apply the method that described conjugate comes cell growth inhibiting and disease therapy, and comprise the test kit of described cytotoxic conjugate.Specifically, Cell binding agent is to identify and in conjunction with the CA6 sugar monoclonal antibody of epi-position or its epitope binding fragments, or its humanization or resurfaced version.

Background technology

Specifically targeted cancerous cells is destroyed to exploitation and do not injure surrounding, the anticancer therapeutic agent of non-cancerous cells and tissue had a lot of trials.The potential very big treatment improving cancer in human patients of such therapeutical agent.

A promising method is that (Sela etc., at Immunoconjugates 189-216 (C.Vogel, ed.1987) for connection Cell binding agent such as monoclonal antibody and cytotoxic drug; Ghose etc., at TargetedDrugs 1-22 (E.Goldberg, ed.1983); Diener etc., at Antibody mediated delivery systems1-23 (J.Rodwell, ed.1988); Pietersz etc., at Antibody mediated delivery systems 25-53 (J.Rodwell, ed.1988); Bumol etc., at Antibody mediated delivery systems 55-79 (J.Rodwell, ed.1988).Depend on the selection to Cell binding agent, based on the expression of molecule expressed on the surface of such cell, these cytotoxic conjugates can be designed as and only to identify and in conjunction with particular type cancer cells.

Cytotoxic drug, such as methotrexate, daunorubicin, Zorubicin, vincristine(VCR), vinealeucoblastine(VLB), melphalan, ametycin and Chlorambucil have been used in such cytotoxic conjugate, and it is connected on various mouse monoclonal antibody.In some cases, drug molecule is connected on antibody molecule by intermediary carrier molecule, and intermediary carrier molecule is serum albumin (Garnett etc., 46Cancer Res.2407-2412 (1986) such as; Ohkawa etc., 23 Cancer Immunol.Immunother. 81-86 (1986); Endo etc., 47Cancer Res.1076-1080 (1980)), dextran (Hurwitz etc., 2 Appl.Biochem.25-35 (1980); Manabi etc., 34 Biochem.Pharmacol.289-291 (1985); Dillman etc., 46 Cancer Res.4886-4891 (1986); Shoval etc., 85 Proc.Natl.Acad.Sci.8276-8280 (1988)) or polyglutamic acid (Tsukada etc., 73 J.Natl.Cane.Inst.721-729 (1984); Kato etc., 27 J.Med.Chem.1602-1607 (1984); Tsukada etc., 52 Br.J.Cancer 111-116 (1985)).

The example having shown the specificity conjugate of some prospects is the conjugate of the DM1 that C242 antibody and maytenin derive, C242 antibody is for CanAg, CanAg is the antigen (Liu etc. expressed on colorectum and pancreatic neoplasm, Proc Natl Acad Sci USA, 93:8618-8623 (1996)).Show the in-vitro evaluation of this conjugate, it is very high to the binding affinity of the CanAg expressed on cell surface, its apparent K _dvalue is 3 × 10 ^-11m, and it is high to the cytotoxic effects of CanAg positive cell, IC ₅₀be 6 × 10 ^-11m.This cytotoxicity is that antigen is dependent, because it is by excessive non-coupled antibody blocking, and because antigen negative cells lowly will be greater than 100 times to the susceptibility of this conjugate.Other example of antibody-DM1 conjugate having high-affinity to respective target cell and have high antigen selecting cell toxicity comprises the conjugate of following material: huN901, and it is the humanization form of anti-human CD56 antibody; HuMy9-6, it is the humanization form of anti-CD 33 antibody; HuC242, it is the humanization form of the antibody of anti-CanAg Muc1 epi-position; HuJ591, it is the disimmunity antibody (deimmunized antibody) of anti-PSMA; Herceptin, it is the humanized antibody of anti-Her2/neu; With than cutting down pearl monoclonal antibody (bivatuzumab), it is the humanized antibody of anti-CD44v6.

Be used for the treatment of in the method for cancer patients updating, other cytotoxic conjugate of exploitation specific recognition particular type cancer cells will be important.

In order to this target, the present invention relates to the exploitation of antibody, described antibody recognition also combines the molecule/acceptor of expressing on cancer cell surfaces, with the exploitation relating to novel cytotoxic conjugate, described conjugate comprises Cell binding agent such as antibody and cytotoxic agent, and described cytotoxic agent special target expresses the molecule/acceptor on cancer cell surfaces.

More specifically, the present invention relates to the sign of new CA6 sialic acid sugar epi-position (sialoglycotope) on the Mucl Saliva Orthana acceptor that is positioned at and expressed by cancer cells, with the preparation relating to antibody, preferred humanized antibody, it identifies the mucinous new CA6 sialic acid sugar epi-position of Mucl, and when there is cytotoxic agent, the growth suppressing the cell of expressing CA6 sugar epi-position can be used to.

Summary of the invention

The present invention includes antibody or its epitope binding fragments, described antibodies specific identification and in conjunction with Mucl Saliva Orthana acceptor novel C A6 sialic acid sugar epi-position.In another embodiment, the present invention includes humanized antibody or its epitope binding fragments, it identifies the novel C A6 sialic acid sugar epi-position (" CA6 sugar epi-position ") of Mucl Saliva Orthana acceptor.

In preferred embodiments, the present invention includes surface reconstruction or the humanization form of mouse-anti-CA6 monoclonal antibody DS6 (" DS6 antibody ") and DS6 antibody, wherein, in light chain and heavy chain, the residue being exposed to surface of antibody or its epitope binding fragments is substituted, to be even more closely similar to known people's antibody surface.Humanized antibody of the present invention and epitope binding fragments thereof have the character of improvement, because compared with mouse form completely, they have less immunogenicity (or right and wrong are immunogenic completely) in its people's object be given.Therefore, the novel sialic acid sugar epi-position on humanization DS6 antibody of the present invention and epitope binding fragments specific recognition Mucl Saliva Orthana acceptor thereof, i.e. CA6 sugar epi-position, and be not immunogenic to people.This humanized antibody and epitope binding fragments thereof can with medicine such as CHROMATOGRAPHIC FRACTIONATION AND MASS (maytansinoid) coupling, by this medicine being oriented to Mucl CA6 sialic acid sugar epi-position, form prodrug antigen-expressing cells to specific cytotoxic.Therefore can use and comprise such antibody and cytotoxic conjugate that is little, high toxic drugs (such as CHROMATOGRAPHIC FRACTIONATION AND MASS, taxanes (taxanes) and CC-1065 analogue) as therapeutical agent, be used for the treatment of tumour, such as mammary gland and ovarian tumor.

The humanization form of DS6 antibody of the present invention is in the following areas in this article by complete characterization: the respective aminoacid sequence of light chain and variable region of heavy chain, the DNA sequence dna of light chain and heavy chain variable region gene, the qualification of complementary determining region (CDRs), the qualification of its surface amino groups acid and disclosing of its method expressed in recombinant form.

In one embodiment, provide humanization DS6 antibody or its epitope binding fragments, it has the heavy chain comprising complementary determining region (CDRs), and described complementary determining region (CDRs) has the aminoacid sequence shown in SEQ ID NOS:1-3.

S Y N M H(SEQ ID NO：1)，

Y I Y P G N G A T N Y N Q K F K G(SEQ ID NO：2)，

G D S V P F A Y(SEQ ID NO：3)，

And there is the light chain comprising complementary determining region (CDRs), described complementary determining region (CDRs) has the aminoacid sequence shown in SEQ ID NOS:4-6:

S A H S S V S F M H(SEQ ID NO：4)，

S T S S L A S(SEQ ID NO：5)，

Q Q R S S F P L T(SEQ ID NO：6)。

Also provide humanization DS6 antibody and epitope binding fragments thereof, it has variable region of light chain, described variable region of light chain has such aminoacid sequence, that is, this sequence and SEQ ID NO:7 or the aminoacid sequence shown in SEQ ID NO:8 have the sequence iden of at least 90%:

QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAGTKLELKR(SEQ ID NO：7)

EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAGTKLELKR(SEQ ID NO：8)。

Also provide humanization DS6 antibody and epitope binding fragments thereof, it has variable region of heavy chain, described variable region of heavy chain has such aminoacid sequence, that is, this sequence and SEQ ID NO:9, SEQ ID NO:10 or the aminoacid sequence shown in SEQ ID NO:11 have the sequence iden of at least 90%:

QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYWGQGTLVTVSA(SEQ ID NO：9)

QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYWGQGTLVTVSA(SEQ ID NO：10)

QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYWGQGTLVTVSA(SEQ ID NO：11)。

In another embodiment, provide humanization DS6 antibody and epitope binding fragments thereof, it has the variable region of light chain of humanization or surface reconstruction, and described variable region of light chain has the aminoacid sequence corresponding to SEQ ID NO:8:

EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAGTKLELKR.(SEQ ID NO：8)。

Similarly, provide humanization DS6 antibody and epitope binding fragments thereof, it has the variable region of heavy chain of humanization or surface reconstruction, and described variable region of heavy chain has the aminoacid sequence corresponding respectively to SEQ ID NO:10 or SEQ ID NO:11:

QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYWGQGTLVTVSA.(SEQ ID NO：10)

QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYWGQGTLVTVSA.(SEQ ID NO：11)。

Humanization DS6 antibody of the present invention and epitope binding fragments thereof also can be included in the replacement in light chain and/or heavy chain amino acid residues, describedly replace on one or more positions of occurring to define with the residue of asterisk in Table 1, the residue of described band asterisk represent be found to be positioned at CDR 5 dusts within, the mouse surface architecture residue (murine surface framework residues) that needs to become people's residue.Such as, the first amino-acid residue Q in mouse sequence (SEQ IDNO:7) is replaced (SEQ ID NO:8), to make antibody humanization by E.But, due to this residue and CDR close, reverse mutation may be needed to be mouse residue Q, to maintain affinity of antibody.

Table 1

This is shown in table 2 further, illustrated therein is people's Variable region surface residues comparison of muDS6 Variable region surface residues and three most homologies.Amino-acid residue in table 1 corresponds to the amino-acid residue with underscore in table 2.

Table 2

Invention further provides cytotoxic conjugate, Cell binding agent that it comprises (1), it identifies and in conjunction with CA6 sugar epi-position, and (2) cytotoxic agent.In cytotoxic conjugate, Cell binding agent has high-affinity to CA6 sugar epi-position, and cytotoxic agent has the cytotoxicity of height to the cell of expressing CA6 sugar epi-position, and therefore cytotoxic conjugate of the present invention defines effective kill agent.

In a preferred embodiment, described Cell binding agent is anti-CA6 antibody or its epitope binding fragments, be more preferably the anti-CA6 antibody of humanization or its epitope binding fragments, wherein cytotoxic agent directly or by the joint that maybe can not cut that can cut is covalently bound to antibody or its epitope binding fragments.In a more preferred embodiment, Cell binding agent is humanization DS6 antibody or its epitope binding fragments, and cytotoxic agent is taxol (taxol), CHROMATOGRAPHIC FRACTIONATION AND MASS, CC-1065 or CC-1065 analogue.

In a preferred embodiment of the present invention, Cell binding agent is the anti-CA6 antibody of humanization, and cytotoxic agent is cytotoxic drug, such as CHROMATOGRAPHIC FRACTIONATION AND MASS or taxanes.

More preferably, Cell binding agent is humanization anti-CA6 antibody DS6, and cytotoxic agent is maytenin compound, such as DM1 or DM4.

The present invention also comprises the method suppressing the Growth of Cells of expressing CA6 sugar epi-position.In preferred embodiments, for suppressing the method for the Growth of Cells of expressing CA6 sugar epi-position to be carried out in vivo, and necrocytosis is caused, although also comprise external and in vitro application.

Present invention provides therapeutic composition, it comprises cytotoxic conjugate and pharmacology acceptable carrier or vehicle.

The present invention comprises the method using described therapeutic composition to treat cancered object further.In preferred embodiments, cytotoxic conjugate comprises anti-CA6 antibody and cytotoxic agent.In a more preferred embodiment, cytotoxic conjugate comprises humanization DS6 antibody-DM1 conjugate, humanization DS6 antibody-DM4 or humanization DS6 antibody-Taxan conjugate, and described conjugate is applied together with pharmacology acceptable carrier or vehicle.

The present invention also comprises test kit, and it comprises anti-CA6 antibody-cell toxic agents conjugate and operation instruction.In preferred embodiments, anti-CA6 antibody is humanization DS6 antibody, and cytotoxic agent is maytenin compound, such as DM1 or DM4, or taxanes, and explanation is to treat cancer stricken object about this conjugate of use.Test kit also comprises for the preparation of the component needed for preparation acceptable on pharmacology, if such as thinner---and conjugate is in lyophilised state or conc forms, and comprises the component for using said preparation.

The present invention also comprises the derivative of the antibody of specific binding and identification CA6 sugar epi-position.In preferred embodiments, by surface reconstruction or humanization in conjunction with the antibody of CA6 sugar epi-position Dispersal risk derivative, wherein derivative has the immunogenicity of reduction to host.

Invention further provides humanized antibody or its fragment, it is labeled further for use in research or diagnostic use.In preferred embodiments, radio-labeling, fluorophore, chromophore, developer or metal ion is labeled as described in.

Also provide diagnostic method, wherein the humanized antibody of described mark or its epitope binding fragments are administered to and suspect cancered object, and measure or the distribution of monitoring mark in subject.

Present invention provides the method by using humanized antibody conjugate of the present invention treatment cancer stricken object, described conjugate or use separately or use with other cytotoxic agent or therapeutic agent.Cancer can be such as following one or more: the sarcoma that mammary cancer, colorectal carcinoma, ovarian cancer, carcinoma of endometrium, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial membrane cancer, carcinoma of the pancreas, wherein CA6 are expressed or cancer or wherein CA6 sugar epi-position are by other cancer to be determined significantly expressed.

Be otherwise noted unless there are other, all reference quoted herein and patent are introduced into herein as a reference.

Accompanying drawing explanation

Fig. 1 shows the result of study of the ability for measuring the cancerous cell line surface selected by DS6 antibodies.By flow cytometry, the fluorescence of the clone that against murine IgG (H+L) two temperature resistance of measurement DS6 primary antibodie and FITC coupling is educated.DS6 antibodies Caov-3 (Figure 1A) and T-47D (Figure 1B) cell, apparent Kd is 1.848nM and 2.586nM respectively.Antigen negative cells system, SK-OV-3 (Fig. 1 C) and Colo205 (Fig. 1 D) display is without antigen specific binding.

Fig. 2 shows the result of the dot blot assay that epi-position is expressed.Caov-3 (Fig. 2 A and Fig. 2 B), SKMEL28 (Fig. 2 C) and Colo205 (Fig. 2 D) cell lysate are put on nitrocellulose filter respectively, then use PRONASE A, Proteinase K, neuraminidase or periodic acid incubation respectively.Then DS6 antibody (Fig. 2 A), CM1 antibody (Fig. 2 B), R24 antibody (Fig. 2 C) or C242 antibody (Fig. 2 D) is used to carry out immunoblotting to this film.

Fig. 3 shows the result of the dot blot assay of DS6 antigen presentation.Caov-3 cell lysate is put on pvdf membrane respectively, then incubation under trifluoromethanesulfonic acid (trifluoromethanesulfoni acid (TFMSA)) existent condition.Then CM1 antibody (1 & 2) or DS6 antibody (3 & 4) is used to carry out immunoblotting to film.

Fig. 4 shows the result of the sugared epitope analysis of DS6 antigen.Will, with N-glycanase (N-glycanase (" N-gly ")), O-glycanase (O-glycanase (" O-gly ")) and/or sialidase (sialidase (" S ")) pretreated Caov-3 lysate point sample to disappearing on nitrocellulose, DS6 antibody or CM1 antibody (Muc-1VNTR) be then used to carry out immunoblotting.

Fig. 5 shows the result of the western blot analysis of DS6 antigen.Cell lysate DS6 antibody carries out immunoprecipitation (immunoprecipitated (" IP ")) and immunoblotting.Antigen corresponds to the protein band being greater than 250kDa observed in antigen positive Caov-3 (Fig. 5 A and Fig. 5 B) and T47D (Fig. 5 C) cell.Antigen negative SK-OV-3 (Fig. 5 D) and Colo205 (Fig. 5 E) clone do not show this band.After immunoprecipitation, use the protein G pearl of (Fig. 5 A) neuraminidase (neuraminidase (" N ")) or (Fig. 5 B) periodic acid (periodic acid (" PA ")) incubation Caov-3 cell lysate.Same gel to make before antibody (" α "), IP merchantable thing (flow through, " FT ") lysate contrast after (" Lys ") and IP run glue.Also use N-glycanase (" N-gly "), O-glycanase (" O-gly ") and/or sialidase (" S ") incubation Caov-3 immunoprecipitate (see Fig. 5 F), wherein detect trace with biotinylation-DS6 and streptavidin-HRP alternatively.

Fig. 6 shows DS6 antibody and the immunoprecipitation of CM1 antibody on Caov-3 (Fig. 6 A) and HeLa (Fig. 6 B) cell lysate and/or the result of immunoblotting.Superposition CM1 and DS6 Western blot signal, shows that DS6 antigen is on Muc1 albumen.In HeLa lysate, Muc1 doublet comes from the expression of Muc1, and this expression is instructed by the not isoallele that repetition number of contacting is different.

Fig. 7 shows the design of DS6 antibody sandwich ELISA (Fig. 7 A) and typical curve (Fig. 7 B).The commercially available CA15-3 standard substance (wherein 1CA15-3 unit=1DS6 unit) of concentration known are used to produce typical curve.

Fig. 8 shows quantitative ELISA typical curve.Use the vitamin H-DS6 of concentration known, itself or caught by the sheep anti-mouse igg (Fig. 8 A) of bed board, or be directly attached to (Fig. 8 B) on ELISA flat board, measure the typical curve (Fig. 8 C) detecting antibody (streptavidin-HRP/ vitamin H-DS6) signal.

Fig. 9 shows the light chain (Fig. 9 A) of mouse DS6 antibody and the cDNA of heavy chain (Fig. 9 B) variable region and aminoacid sequence.Three CDR in each sequence are by underscore (Kabat definition) in addition.

Figure 10 shows the light chain (Figure 10 A) and heavy chain (Figure 10 B) CDRs that define the mouse DS6 antibody determined according to Kabat.Software is built for heavy chain CDRs, AbM mould and produces slightly different definition (Figure 10 C).

Figure 11 shows light chain (" muDS6LC ") (the 1-95 residue of SEQ ID NO:7) and heavy chain (" muDS6HC ") (the residue 1-98 of the SEQ ID NO:9) aminoacid sequence of mouse DS6 antibody, and the germ line sequences of they and IgV κ ap4 (SEQ ID NO:23) and IgVh J558.41 (SEQ ID NO:24) gene is compared.Grey represents sequence difference.

Figure 12 shows 10 light chains and heavy chain antibody sequence in Brookhaven database with analytic structure (solved structure) file, they and mouse DS6 (muDS6) light chain (" muDS6LC ") and the most homology of heavy chain (" muDS6HC ") sequence.The minimum order collating sequence of homology is up to homology.

Figure 13 show for predict which framework residue of mouse DS6 antibody chain variable region be surface can and surperficial accessibility (surface accessibility) data and calculating.Does is the position with the average surface accessibility of 25-35% labeled (*? *), second is carried out to them and take turns analysis.DS6 antibody chain variable region (Figure 13 A) and variable region of heavy chain (Figure 13 B).

Figure 14 shows prDS6 v1-0 Mammalian expression plasmid figure.It is chimeric with humanized DS6 antibody that this plasmid is used to set up and express restructuring.

Figure 15 shows the aminoacid sequence of mouse (" muDS6 ") and humanization (" huDS6 ") (v1.0 & v1.2) DS6 light chain of antibody (Figure 15 A) and heavy chain (Figure 15 B) variable knob domain.

Figure 16 shows cDNA and the aminoacid sequence of the variable region of light chain of humanization DS6 antibody (" huDS6 ") (1.01 and 1.21).

Figure 17 shows cDNA and the aminoacid sequence of the variable region of heavy chain of humanization DS6 antibody 1.01 (Figure 17 A) and 1.21 (Figure 17 B).

Figure 18 shows the mouse DS6 (muDS6) of the analysis that comfortable KB cell carries out, chimeric DS6 (chDS6), and the flow cytometry binding curve of people DS6 form 1.01 (huDS6v1.01) and huDS6 form 1.21 (huDS6v1.21).Murine antibody, chimeric antibody are similar with the avidity (muDS6=0.82nM, chDS6=0.69nM, huDS6v1.01=0.82nM, huDS6v1.21=0.85nM) of v1.21DS6 antibody with people v1.01, and this shows that surface reconstruction does not reduce avidity.

Figure 19 shows the result of the Competition binding assay of muDS6, chDS6, huDS6 v1.01 and huDS6 v1.21 antibody and biotinylation muDS6.By naked muDS6, chDS6, huDS6v1.01 of various concentration and huDS6v1.21 and 2nM vitamin H-muDS6 and the mixing of streptavidin-DTAF second class grade chemical.The IC50 (muDS6=1.9nM, chDS6=1.7nM, huDS6v1.01=3.0nM and huDS6v1.21=1.9nM) of all antibody is similar, and this shows that humanization does not reduce avidity.

Figure 20 shows the measurement result of the DS6 antibody of non-coupling and the binding affinity of DS6 antibody-DM1 conjugate.Result shows, and DM1 coupling does not adversely affect the binding affinity of antibody.Apparent Kd (3.902nM) (" DS6-DM1 ") the more naked antibody (2.020nM) (" DS6 ") of DS6 antibody-DM1 conjugate is slightly large.

Figure 21 shows when against murine IgG (H+L) DM1 conjugate (2 ° of Ab-DM1) exists or lack, the result that the indirect cell survival rate using DS6 antibody to carry out measures.Antigen positive Caov-3 cell to be only killed (IC in DS6 antibody dependent mode when there is secondary conjugate (secondary conjugate) (" DS6+2 ° of Ab-DM1 ") ₅₀=424.9pM).

The result that the CDC (CDC) that Figure 22 shows muDS6 antibody is tested.Result shows, on HPAC (Figure 22 A) and ZR-75-1 (Figure 22 B) cell, there is not the effect of the CDC mediation of DS6 antibody.

Figure 23 shows the result of DS6 antibody-DM1 conjugate and free maytenin cytotoxic assay in vitro.In colony formation test (clonogenic assay), DS6 antigen positive ovary (Figure 23 A), mammary gland (Figure 23 B), uterine cervix (Figure 23 C) and pancreas (Figure 23 D) cancerous cell line are exposed to DS6 antibody-DM1 conjugate continuously, measure cytotoxicity (left figure).Be exposed to free maytenin by 72h, determine the maytenin susceptibility (right figure) of these clones similarly.Tested ovarian cancer cell line is OVCAR5, TOV-21G, Caov-4 and Caov-3.Tested breast cancer cell line is T47D, BT-20 and BT-483.Tested cervical cancer cell system is KB, HeLa and WISH.Tested pancreatic cancer cell is HPAC, Hs766T and HPAF-11.

Figure 24 shows DS6 antibody-DM1 conjugate cell toxicity test result in vitro.In MTT cell survival rate is analyzed, people's ovary (Figure 24 A, Figure 24 B and Figure 24 C), mammary gland (Figure 24 D and Figure 24 E), uterine cervix (Figure 24 F and Figure 24 G) and pancreas (Figure 24 H and Figure 24 I) cancer cells are killed in the mode depending on DS6 antibody-DM1 conjugate.Naked DS6 does not adversely affect the growth of these cells, and this shows that DM1 coupling is needs for cytotoxicity.

Figure 25 A shows the anti-tumor in vivo efficacy study result of DS6 antibody-DM1 conjugate to the subcutaneous KB tumor xenogeneic graft set up.At the 0th day inoculated tumour cell, gave first time treatment at the 6th day.Continuous every day carries out immune conjugate treatment, amounts to 5 dosage.Once gross tumor volume is more than 1500mm ³, then PBS control animal is put to death.Conjugate is used, respectively the antibody concentration of correspondence 5.7 and 8.5mg/kg with the dosage of 150 or 225 μ g/kg DM1.The body weight (Figure 25 B) of mouse is monitored during studying.

Figure 26 shows DS6 antibody-DM1 conjugate to the antitumor efficacy result of study of the Subcutaneous tumor heterograft of having set up.At the 0th day inoculation OVCAR5 (Figure 26 A and Figure 26 B), TOV-21G (Figure 26 C and Figure 26 D), HPAC (Figure 26 E and Figure 26 F) and HeLa (Figure 26 G and Figure 26 H) cell, give immune conjugate treatment the 6th day and the 13rd day.Once gross tumor volume is more than 1000mm ³, then PBS control animal is put to death.Conjugate is used, corresponding to the antibody concentration of 27.7mg/kg with the dosage of 600 μ g/kg DM1.Gross tumor volume (Figure 26 A, Figure 26 C, Figure 26 E and 26G) and the body weight (Figure 26 B, Figure 26 D, Figure 26 F and Figure 26 H) of mouse is monitored during studying.

Figure 27 shows DS6 antibody-DM1 conjugate to effect result of study in the body of intraperitoneal OVCAR5 tumour.At the 0th day peritoneal injection tumour cell, give immune conjugate treatment the 6th day and the 13rd day.Once body weight loss is more than 20%, then put to death animal.

The flow cytometry binding curve that binding affinity study of DS6 antibody on HeLa cell that Figure 28 shows naked DS6 antibody and Taxan coupling obtains.Taxan (MM1-202) coupling does not adversely affect the binding affinity of antibody.The apparent Kd (1.24nM) of DS6-MM1-202 conjugate is slightly larger than naked DS6 antibody (620pM).

Figure 29 shows external combination and the effect of the 1.01 form antibody coupling matters of humanization DS6.HuDS6v1.01 and DM4 coupling does not almost affect (Figure 29 A) for the avidity of huDS6v1.01 and KB cell.HuDS6v1.01-DM4 expresses WISH cell to DS6 and shows strong vitro cytotoxicity, IC ₅₀0.44nM (Figure 29 B).

Figure 30 shows the vivo effect result of study of huDS6v1.01-DM4 conjugate in HPAC pancreatic cancer cell model.HuDS6v1.01-DM4 shows effective antitumour activity, and the B4-DM4 contrast conjugate that its target is not expressed in HPAC model there is no activity (Figure 30 A).The dosage of 200 μ g/kg does not have toxicity to animal, if there is not (Figure 30 B) as indicated in body weight loss.

Detailed Description Of The Invention

The invention provides the fragment of anti-CA6 monoclonal antibody, anti-CA6 humanized antibody and anti-CA6 antibody, and further feature.Each antibody of the present invention and antibody fragment are designed to specific recognition and are combined in the CA6 sugar epi-position on cell surface.Known CA6 is expressed by many human tumors: the serous ovarian cancer of 95%, the endometrial ovarian cancer (endometrioid ovarian carcinomas) of 50%, 50% tumor of cervix, 69% adenomyoma (neoplasms of the endometrius), 80% vulva knurl, 60% breast cancer, 67% Vipoma and 48% urothelium knurl (tumors of the urothelium), but it is seldom expressed by Normal human tissue.

The people Int.J.Cancer such as Kearse 88 (6): 866-872 (2000) report, when they use doma supernatant to identify, the albumen finding that there is CA6 epi-position is above accredited as mistakenly the 80kDa albumen with the N connection sugar comprising CA6 epi-position.By using the DS6 of purifying, we have demonstrated CA6 epi-position and have been found to be positioned on the O connection sugar of the non-disulfide linkage connection glycoprotein of more than 250kDa.In addition, this glycoprotein through being accredited as Saliva Orthana, Muc1.Because different Muc1 allelotrope repeats to have the different tandem repeat sequence of number in (VNTR) structural domain at different number series winding, therefore cell often expresses different Muc1 albumen (Taylor-Papadimitriou, Biochim.Biophys.Acta 1455 (2-3): 301-13 (1999) of two kinds of different sizes.Because the repetition number in VNTR structural domain is different and the difference of glycosylation, the molecular weight of Muc1 changes with clone.

The susceptibility of CA6 immunoreactivity to periodic acid shows, CA6 is carbohydrate epitope " sugared epi-position (glycotope) ".CA6 immunoreactivity shows by the other susceptibility of the neuraminic acid ferment treatment from vibrio cholerae (Vibrio cholera), CA6 epi-position is sialic acid dependency sugar epi-position, is therefore " sialic acid sugar epi-position (sialoglycotope) ".

The sign detailed content of CA6 (consulting following) can be found in example 2.At WO 02/16401; Wennerberg etc., Am.J.Pathol.143 (4): 1050-1054 (1993); Smith etc., Human Antibodies9:61-65 (1999); Kearse etc., Int.J.Cancer 88 (6): 866-872 (2000); Smith etc., Int.J.Gynecol.Pathol.20 (3): 260-6 (2001); With in Smith etc., Appl.Immunohistochem.Mol.Morphol.10 (2): 152-8 (2002), the other detailed content about CA6 can be found.

The present invention also comprises the cytotoxic conjugate containing two kinds of main ingredients.First component is Cell binding agent, its identify and in conjunction with CA6 sugar epi-position.Described Cell binding agent should with the CA6 sialic acid sugar epi-position of the specific recognition of height on Muc 1, so that described cytotoxic conjugate only identifies and cell desired by combining.The specificity of height allows this conjugate to play a role in a targeted manner, does not almost have the side effect because non-specific binding causes.

In another embodiment, Cell binding agent of the present invention is also with the avidity identification CA6 of height sugar epi-position, so that this conjugate will contact time enough with target cell, thus the cytotoxic drug part of conjugate is worked on cell, and/or conjugate is made to have time enough by cell internalizing.

In a preferred embodiment, cytotoxic conjugate comprises anti-CA6 antibody as Cell binding agent, is more preferably the anti-CA6 monoclonal antibody of mouse DS6.In a preferred embodiment, cytotoxic conjugate comprises humanization DS6 antibody or its epitope binding fragments.Cytotoxic agent with the specific recognition CA6 of height, and is guided to abnormal cells or tissue, such as cancer cells by DS6 antibody capable in a targeted manner.

The second component of cytotoxic conjugate of the present invention is cytotoxic agent.In preferred embodiments, cytotoxic agent is taxol, CHROMATOGRAPHIC FRACTIONATION AND MASS such as DM1 or DM4, CC-1065 or CC-1065 analogue.In preferred embodiments, Cell binding agent of the present invention directly or by cutting maybe can not cut joint and cytotoxic agent is covalently bound.

Cell binding agent, cytotoxic agent and joint are discussed hereinafter in more detail.

cell binding agent

Compound of the present invention depends on the careful selection to suitable Cell binding agent as the validity of therapeutical agent.Cell binding agent can be at present known or will be known any kind, comprise peptide and non-peptide class.Cell binding agent can be can with specificity or the non specific manner any compound in conjunction with cell.Generally speaking, these Cell binding agent can be antibody (particularly monoclonal antibody), lymphokine, hormone, somatomedin, VITAMIN, the nutrient transport factor (such as Transferrins,iron complexes) or other cell-binding molecules any or material.

Can be comprised by the example more specifically of the Cell binding agent used:

(a) polyclonal antibody;

(b) monoclonal antibody;

(c) antibody fragment, such as Fab, Fab ' and F (ab ') ₂, Fv (Parham, J.Immunol.131:2895-2902 (1983); Spring etc., J Immunol.113:470-478 (1974); Nisonoff etc., Arch.Biochem.Biophys.89:230-244 (1960));

(d) Interferon, rabbit (such as α, β, γ);

(e) lymphokine, such as IL-2, IL-3, IL-4, IL-6;

(f) hormone, such as Regular Insulin, TRH (thyrotrophin-releasing hormone), MSH (melanotropin), steroid hormone, such as male sex hormone and oestrogenic hormon;

(g) somatomedin and G CFS, such as EGF, TGF-α, FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158 (1984));

(h) Transferrins,iron complexes (O ' Keefe etc., J.Biol.Chem.260:932-937 (1985)); With

(i) VITAMIN, such as folic acid.

antibody

Select the Cell binding agent be applicable to be the key selected, this depends on the concrete cell mass for being targeted, but generally speaking, maybe can be produced, then preferred antibody, more preferably monoclonal antibody if the antibody be applicable to can be utilized.

Monoclonal antibody technique allows to produce extremely specific Cell binding agent with the form of monoclonal antibody specific.By with the such as complete target cell of interested antigen, from target cell, isolated antigen, totivirus, attenuation totivirus and viral protein such as viral capsid proteins immune mouse, rat, hamster or other Mammals any and the technology producing monoclonal antibody are known especially in this area.Also people's cell of sensitization can be used.Producing the another kind of method of monoclonal antibody is the phage library using scFv (single-chain variable), especially people scFv (see such as, Griffiths etc., United States Patent (USP) 5,885,793 and 5,969,108; McCafferty etc., WO92/01047; Liming etc., WO 99/06587).

Typical antibody is made up of the light chain that two that are connected by disulfide linkage identical heavy chains are identical with two.Variable region is a part for heavy chain of antibody and light chain, and its sequence between antibody is different, and it acts synergistically in each specific antibodies is to the associativity of its antigen and specificity.Mutability is not equally distributed in whole antibody variable region usually.Within three fragments that it typically concentrates on variable region, it is referred to as complementarity-determining region (CDRs) or hypervariable region, is positioned at light chain and variable region of heavy chain.The part in variable region with higher conservative property is referred to as skeleton district.The variable region of heavy chain and light chain comprises four skeleton districts, mainly adopts β-sheet configuration, and wherein each skeleton district is connected by three CDRs, and described three CDRs form the ring connecting β-chip architecture, in some cases, forms a part for β-chip architecture.CDRs in each chain is maintained at position close to each other by skeleton district, and the antigen binding site (E.A.Kabat etc. forming antibody are helped together with the CDRs from another chain, Sequences of Proteins of Immunological Interest, Fifth Edition, 1991, NIH).

Constant region is a part for heavy chain.Although do not participate in the combination of antibody and antigen directly, but it shows various effector functions really, and such as antibody participates in antibody dependent cellular cytotoxicity.

Applicable monoclonal antibody used in this invention comprises mouse DS6 monoclonal antibody (United States Patent (USP) 6,596,503; ATCC preserving number PTA-4449).

the DS6 antibody of humanization or surface reconstruction

Preferably, humanization anti-CA6 antibody is used as Cell binding agent of the present invention.The preferred embodiment of such humanized antibody is humanization DS6 antibody, or its epitope binding fragments.

Humanized target reduces the immunogenicity being incorporated into the heteroantibody such as murine antibody of human body, keeps whole antigen-binding affinity and the specificity of this antibody simultaneously.

Use several technology can generating humanized antibodies, such as surface reconstruction (resurfacing) and CDR transplant (CDR grafting).As utilized herein, resurfacing techniques employs the combination of Molecular modeling, statistical study and mutagenesis, changes the non-CDR surface of antibody variable region, with the surface of the known antibodies of simulation target host.

At United States Patent (USP) 5,639, disclose the strategy for the surface reconstruction of antibody and method in 641 (Pedersen etc.), and for reducing antibody other method immunogenic in different hosts, this is incorporated herein its full content as a reference.In brief, in a preferred method, (1) produce the position comparison of a series of heavy chain of antibody and variable region of light chain, to provide one group of position exposed at heavy chain and variable region of light chain skeleton surface, wherein the comparison position of all variable regions has at least about 98% to be identical; (2) for rodent antibodies (or its fragment) determines one group of amino-acid residue exposed at heavy chain and variable region of light chain skeleton surface; (3) identify one group of amino-acid residue exposed at heavy chain and variable region of light chain skeleton surface, it is the most identical with described rodent surface exposed amino acid residue group; (4) be used in that group heavy chain of qualification in step (3) and variable region of light chain skeleton surface to expose amino-acid residue and be substituted in the amino-acid residue that group heavy chain of definition in step (2) and variable region of light chain skeleton surface expose, except those are positioned at any atom of any residue of the complementarity-determining region of rodent antibodies within amino-acid residue; (5) Humanized rodent antibodies with binding specificity is produced.

Other technology multiple can be used to carry out humanized antibody, comprise CDR-and transplant (EP 0 239 400; WO91/09967; United States Patent (USP) 5,530,101; With 5,585,089), veneer (veneering) or surface reconstruction (EP0 592 106; EP 0 519 596; Padlan E.A., 1991, Molecular Immunology 28 (4/5): 489-498; Studnicka G.M. etc., 1994, Protein Engineering 7 (6): 805-814; Roguska M.A. etc., 1994, PNAS 91:969-973) and chain rearrangement (United States Patent (USP) 5,565,332).People's antibody can be prepared by multiple techniques known in the art, comprise phage display method.Also see United States Patent (USP) 4,444,887,4,716,111,5,545,806 and 5,814,318; With International Patent Application Publication No. WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 (full content of described document is introduced into as a reference).

In preferred embodiments, the invention provides humanized antibody or its fragment of the new sialic acid sugar epi-position (CA6 sugar epi-position) identified on Muc1 Saliva Orthana.In another embodiment, humanized antibody or its epitope binding fragments have the extra ability suppressing the Growth of Cells of expressing CA6 sugar epi-position.

In a more preferred embodiment, provide surface reconstruction or the humanization form of DS6 antibody, wherein in light chain and heavy chain, the residue that the surface of antibody or its fragment exposes is substituted, to be more similar to known people's antibody surface.Humanization DS6 antibody of the present invention or its epitope binding fragments have the characteristic of improvement.Such as, humanization DS6 antibody or its epitope binding fragments identify the new sialic acid sugar epi-position (CA6 sugar epi-position) on Muc1 Saliva Orthana specifically.More preferably, humanization DS6 antibody or its epitope binding fragments have the other ability suppressing the Growth of Cells of expressing CA6 sugar epi-position.Humanized antibody or its epitope binding fragments can be coupled to medicine such as CHROMATOGRAPHIC FRACTIONATION AND MASS, by by drug targeting in new Muc1 sialic acid sugar epi-position CA6, form prodrug antigen-expressing cells to SC.Comprise such antibody and cytotoxic conjugate that is little, high toxic drugs (such as CHROMATOGRAPHIC FRACTIONATION AND MASS, taxanes and CC-1065 analogue) can be used as treating tumour, the therapeutical agent of such as mammary tumor and ovarian tumor.

The humanization form of DS6 antibody is also fully characterized in this article in following: the DNA sequence dna of its light chain and variable region of heavy chain aminoacid sequence separately, light chain and heavy chain variable region gene, the qualification of CDRs, the qualification of its surface amino groups acid and disclosing the method that it is expressed in recombinant form.

In one embodiment, provide humanized antibody or its epitope binding fragments, it has the heavy chain comprising CDRs, and described CDRs has by the aminoacid sequence shown in SEQ ID NOs:1-3:

S Y N M H(SEQ ID NO：1)

Y I Y P G N G A T N Y N Q K F K G(SEQ ID NO：2)

G D S V P F A Y(SEQ ID NO：3)

When heavy chain CDRs is measured by AbM modeling software, it is represented by SEQ ID NOs:20-22:

G Y T F T S Y N M H(SEQ ID NO：20)

Y I Y P G N G A T N(SEQ ID NO：21)

G D S V P F A Y(SEQ ID NO：22)

In identical embodiment, humanized antibody or its epitope binding fragments have the light chain comprising CDRs, and described CDRs has the aminoacid sequence shown in SEQ ID NOS:4-6:

S A H S S V S F M H(SEQ ID NO：4)

S T S S L A S(SEQ ID NO：5)

Q Q R S S F P L T(SEQ ID NO：6)

Also provide the humanized antibody and epitope binding fragments thereof with variable region of light chain, the aminoacid sequence that described variable region of light chain has, the aminoacid sequence represented with SEQ ID NO:7 or SEQ ID NO:8 shares the sequence iden of at least 90%:

Similarly, provide the humanized antibody and epitope binding fragments thereof with variable region of heavy chain, the aminoacid sequence that described variable region of heavy chain has, share the sequence iden of at least 90% with the aminoacid sequence shown in SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11:

In another embodiment, provide humanized antibody and the epitope binding fragments thereof of the variable region of light chain with humanization or surface reconstruction, described variable region has the aminoacid sequence corresponding to SEQ ID NO:8:

Similarly, provide humanized antibody and the epitope binding fragments thereof of the variable region of heavy chain with humanization or surface reconstruction, described variable region has the aminoacid sequence corresponding to SEQ ID NO:10 or SEQ ID NO:11:

Humanized antibody of the present invention and epitope binding fragments thereof also can comprise following light chain and/or the form of variable region of heavy chain, namely, wherein close to CDRs people's surface amino groups acid residue is replaced by corresponding muDS6 surface residue, described replacement occurs in one or more positions that the residue of asterisk mark limits in table 1 (Kabat numbering), and object is the binding affinity and the specificity that keep muDS6.

Table 1

Original amino acid and the DNA sequence dna of DS6 light chain of antibody and heavy chain are disclosed herein, and humanization form.But scope of the present invention is not limited to the antibody and the fragment that comprise these sequences.On the contrary, be combined as all antibody and the fragment of the CA6 of tumour-specific sugar epi-position unique on Muc 1 acceptor specifically, all comprise in the present invention.Preferably, the biological activity of the antibody of specific binding CA6 and fragment also antagonism acceptor.More preferably, such antibody there is no agonist activity further.Therefore, antibody of the present invention and antibody fragment can be different from DS6 antibody or its humanized derivative thereof on the aminoacid sequence of its framework, mutual not determining area (CDRs) and/or light chain and heavy chain, but still within the scope of the present invention.

The CDRs of DS6 antibody is identified by modeling, and its molecular structure is predicted.In addition, although mutual not determining area (CDRs) is important for epi-position identification, but they are not required to antibody of the present invention and fragment.Therefore, provide the antibody and fragment that have and improve characteristic, it passes through affinity maturation of such as antibody of the present invention and produces.

The mouse light chain IgV κ ap4 germline gene and the heavy chain IgVh J558.41 germline gene that probably derive DS6 are shown in Figure 11, and the sequence of itself and DS6 antibody is compared.The somatic mutation possible in DS6 antibody of this Identification, is included in mutually the several somatic mutations in not determining area (CDRs).

The heavy chain of DS6 antibody and the sequence of variable region of light chain, and the sequence of the CDRs of DS6 antibody is not known in the past, it is illustrated in figures 9 a and 9b.Such information can be used to the DS6 antibody of generating humanized form.

antibody fragment

Antibody of the present invention comprises above-mentioned disclosed full length antibody and epitope binding fragments.As used herein, " antibody fragment " comprises any part of antibody, and this part remains the ability combining the epi-position identified by full length antibody, is generally known as " epitope binding fragments ".The example of antibody fragment includes but not limited to Fab, Fab ' and F (ab ') ₂, Fd, scFv s (scFv), single-chain antibody, two sulphur connect Fvs (dsFv) and comprise V _lor V _hthe fragment in district.The epitope binding fragments comprising single-chain antibody can comprise separately variable region or with the complete area in following region or subregion combined: hinge area, C _h1, C _h2 and C _h3 structural domains.

Such fragment can comprise one or two Fab fragment or F (ab ') ₂fragment.Preferably, antibody fragment comprises all six CDRs of whole antibody, although comprise the region in below all such regions, such as the fragment of three, four or five CDRs also has function.In addition, fragment can be any one maybe can combining following immunoglobulin class: IgG, IgM, IgA, IgD or IgE and subclass thereof.

By using enzyme such as papoid (Fab fragment) or stomach en-(F (ab ') ₂fragment), through proteolytic cleavage, Fab and F (ab ') can be produced ₂fragment.

Strand FVs (scFvs) fragment is epitope binding fragments, and it comprises and antibody chain variable region (V _l) at least one fragment connect antibody heavy chain variable region (V _h) at least one fragment.Joint can be short flexible peptide, and it is selected in order to ensure once (V _l) and (V _h) region is when being connected, and appropriate three dimensional fold can occur, to keep the target molecule binding specificity deriving the complete antibody of this single chain antibody fragments.(V _l) or (V _h) C-terminal of sequence can by joint and complementary (V _l) or (V _h) amino-terminal end of sequence is covalently bound.

Single chain antibody fragments of the present invention comprises such aminoacid sequence, this aminoacid sequence has at least one in the variable region of in this manual described complete antibody or complementarity-determining region (CDRs), but lacks the some or all of constant domain of those antibody.These constant domain to antigen in conjunction with optional, but constitute the major portion of complete antibody structure.Therefore single chain antibody fragments can overcome and comprises some relevant problems of the antibody of some or all constant domain with using.Such as, often there is not the less desirable interaction between biomolecules and CH in single chain antibody fragments, or other less desirable biological activity.In addition, single chain antibody fragments is more much smaller than complete antibody, therefore can have larger capillary permeability than complete antibody, and this makes single chain antibody fragments more effectively to locate and in conjunction with target antigen combining site.And, in prokaryotic cell prokaryocyte, antibody fragment can be produced with relatively large scale, thus be convenient to its production.In addition, the relatively little size of single chain antibody fragments makes it less compared with complete antibody possibility of challenge in acceptor.

Molecular cloning, antibody phage display library or similar technique known by the technical staff can be passed through and produce single chain antibody fragments.Such as, at eukaryotic cell or prokaryotic cell prokaryocyte, can comprise in bacterium and prepare these protein.Be used in various phage display method known in the art and also can produce epitope binding fragments of the present invention.In phage display method, function antibody structural domain is illustrated in the surface of the phage particle carrying their polynucleotide sequence of coding.Especially, such phage can be used to show the epi-position binding domains of being expressed by natural origin library (repertoire) or combinatorial antibody library (such as people or mouse).Can express in conjunction with the phage of the epi-position binding domains of interested antigen with antigen selection or qualification, such as, use combined or be captured to the labelled antigen of solid surface or pearl.The phage used in these methods is generally filobactivirus, comprise fd and M13, binding domains by phage expression, on the gene III that Fab, Fv or the stable Fv antibody domain of disulfide linkage are fused to phage by recombination method or gene VIII protein.

The example that can be used to the phage display method preparing epitope binding fragments of the present invention comprises those at Brinkman etc., 1995, J.Immunol.Methods 182:41-50; Ames etc., 1995, J.Immunol.Methods 184:177-186; Kettleborough etc., 1994, Eur.J.Immunol.24:952-958; Persic etc. 1997, Gene 187:9-18; Burton etc., 1994, Advances in Immunology 57:191-280; PCT application PCT/GB91/01134; The open WO 90/02809 of PCT; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; With United States Patent (USP) 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969, method disclosed in 108, is incorporated herein the full content of each document as a reference.

After phage is selected, encode fragment phage part can separated and use, epitope binding fragments is produced by carrying out expression in the host selected, host comprises mammalian cell, insect cell, vegetable cell, yeast and bacterium, this uses recombinant DNA technology to carry out, such as, hereafter describe in detail.Such as, restructuring also can be utilized to produce Fab, Fab ' and F (ab ') ₂the technology of fragment, is used in methods known in the art and carries out, and such as those are disclosed in PCT publication number WO 92/22324; Mullinax etc., 1992, BioTechniques 12 (6): 864-869; Sawai etc., 1995, AJRI 34:26-34 and Better etc., the method in 1988, Science 240:1041-1043; The full content of above-mentioned reference is introduced into as a reference.The example that can be used to the technology producing scFv s and antibody comprises those at United States Patent (USP) 4,946,778 and 5,258,498; Huston etc., 1991, Methods in Enzymology 203:46-88; Shu etc., 1993, PNAS 90:7995-7999; Technology described in Skerra etc., 1988, Science 240:1038-1040.

function equivalent

The function equivalent of anti-CA6 antibody and humanization anti-CA6 antibody is also included within scope of the present invention.Term " function equivalent (functional equivalents) " comprise such as there is homologous sequence antibody, chimeric antibody, artificial antibody and modified antibodies, wherein each function equivalent is defined by its ability in conjunction with CA6.Technician will understand, and exist overlapping in the molecular population being referred to as " antibody fragment " and the colony being referred to as " function equivalent ".Produce the method for function equivalent such as at PCT application WO 93/21319, european patent application 239,400; PCT application WO 89/09622; European patent application 338,745 and European patent application EP 332, be disclosed in 424, and its respective full content is introduced into as a reference.

The antibody with homologous sequence is those antibody with following aminoacid sequence, and the aminoacid sequence of described aminoacid sequence and anti-CA6 antibody of the present invention and the anti-CA6 antibody of humanization has sequence homology.Preferably, homology is the amino acid sequence homologous with the variable region of anti-CA6 antibody of the present invention and the anti-CA6 antibody of humanization." sequence homology " that be applied to aminoacid sequence is herein defined as, a sequence and another aminoacid sequence have at least about sequence homology of 90%, 91%, 92%, 93% or 94%, more preferably there is at least about sequence homology of 95%, 96%, 97%, 98% or 99%, such as, according to Pearsonand Lipman, FASTA searching method in Proc.Natl.Acad.Sci.USA 85,2444-2448 (1988) measures.

As used herein, chimeric antibody is the antibody of different piece derived from different animals kind of wherein antibody.Such as, have and match derived from the antibody of the variable region of mouse monoclonal antibody and human normal immunoglobulin constant region.The method producing chimeric antibody is known in the art.See such as Morrison, 1985, Science 229:1202; Oi etc., 1986, BioTechniques 4:214; Gillies etc., 1989, J.Immunol.Methods 125:191-202; United States Patent (USP) 5,807,715; 4,816,567 and 4,816,397, be incorporated herein its full content as a reference.

By being substituted in human skeleton's structural domain the complementary determining region of such as murine antibody, prepare the humanization form of chimeric antibody, for example, see PCT publication number WO 92/22653.Humanized chimeric antibody preferably has constant region and the variable region of the complementary determining region (CDRs) being different from substantially or exclusively deriving from corresponding human antibody regions, and substantially or exclusively derives from the complementary determining region (CDRs) of non-human mammal.

Artificial antibody comprises scFv fragment, double antibody, three chain antibodies, four chain antibodies and atom (mru) (review see Winter, G.and Milstein, C., 1991, Nature 349:293-299; Hudson, P.J., 1999, Current Opinion in Immunology 11:548-557), wherein each has antigen binding capacity.In Single-Chain Fv Fragment of Murine (scFv), the V of antibody _hand V _lstructural domain is connected by flexible peptide.Typically, this joint peptide is the length of about 15 amino-acid residues.If joint is less, such as 5 amino acid, then form double antibody, and it is divalence scFv dimer.If joint is reduced to below three amino-acid residues, then form three poly structures and four poly structures that are referred to as three chain antibodies and four chain antibodies.The minimum combining unit of antibody is CDR, and typically be the CDR2 of heavy chain, it has specific recognition and binding ability, can be used separately.Such fragment is referred to as molecular recognition unit (molecular recognition unit) or atom (mru).With short joint peptide, several such atom (mru) can be linked together, thus form made binding proteins's matter compared with single atom (mru) with more high affinity.

The function equivalent of the application also comprises the antibody of modification, such as, by the antibody of the molecule of any type and the covalently bound modified of antibody.Such as, the antibody of modification comprises such as by glycosylation, acetylize, Pegylation, phosphorylation, amidation, cut by known protection/blocking groups derivatize, proteolysis, be connected to cell ligand or other albumen etc. and adorned antibody.The covalently bound antibody that do not stop produces anti-idiotype reaction.Can be undertaken these by known technology to modify, include but not limited to the metabolism synthesis etc. of specific chemical cutting, acetylize, formylation, tunicamycin.In addition, the antibody of modification can comprise one or more atypical amino acids.

By exchanging the different CDRs on different chain in different frames, function equivalent can be produced.Therefore, such as, substituting by different heavy chains, for one group of specific CDRs, different classes of antibody is possible, such as, can produce IgG1-4, IgM, IgA1-2, IgD, IgE antibody type and isotype thus.Similarly, by being embedded in one group of given CDRs at the framework synthesized completely, the artificial antibody be included within the scope of the invention can be produced.

Being used in a lot of method known in the art, by carrying out suddenling change, lack and/or inserting at the variable and/or constant-region sequences being positioned at one group of specific CDRs flank, easily can producing function equivalent.

Antibody fragment of the present invention and function equivalent comprise those molecules, and it compares with DS6 antibody, with detectable degree in conjunction with CA6.Detectable combination degree comprises all values within least 10% to 100% scope of the binding ability of mouse DS6 antibody and CA6, and preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99%.

the antibody improved

CDRs is most important for epi-position identification and antibodies.But, can the residue forming CDRs be changed, and do not disturb antibody recognition to associate the ability of epi-position with in conjunction with it.Such as can carry out such change, it does not affect epi-position identification, but increases antibody to the binding affinity of epi-position.

Therefore, the form of the improvement of mouse and humanized antibody is also included within scope of the present invention, described form also specific recognition in conjunction with CA6, preferably have the avidity of increase.

Several researchs, based on the understanding to primary antibody sequence, have been investigated and introduced impact (Yang, the W.P. etc. of the change of one or more amino acid on its characteristic such as keying action and expression level on the various positions of antibody sequence, 1995, J.Mol.Biol., 254,392-403; Rader, C. etc., 1998, Proc.Natl.Acad.Sci.USA, 95,8910-8915; Vaughan, T.J. etc., 1998, Nature Biotechnology, 16,535-539).

In these researchs, such as oligonucleotide mediated site-directed mutagenesis, cassette mutagenesis, fallibility PCR, DNA is used to reset or these methods of colibacillary mutator, by the heavy chain in change CDR1, CDR2, CDR3 or skeleton district and light chain gene sequence, create the equivalent (Vaughan of initial antibody, T.J. etc., 1998, Nature Biotechnology, 16,535-539; Adey, N.B. etc., 1996, Chapter 16, pp.277-291, at " Phage Display of Peptides and Proteins ", Eds.Kay, B.K. etc., AcademicPress).These methods changing the sequence of initial antibody have caused the avidity of secondary antibodies to improve (Gram, H. etc., 1992, Proc.Natl.Acad.Sci.USA, 89,3576-3580; Boder, E.T. etc., 2000, Proc.Natl.Acad.Sci.USA, 97,10701-10705; Davies, J.and Riechmann, L., 1996, Immunotechnolgy, 2,169-179; Thompson, J. etc., 1996, J.Mol.Biol., 256,77-88; Short, M.K. etc., 2002, J.Biol.Chem., 277,16365-16370; Furukawa, K. etc., 2001, J.Biol.Chem., 276,27622-27628).

By changing the similar directional strategy of one or more amino-acid residues of antibody, the antibody sequence described in the present invention can be used to develop the anti-CA6 antibody having and improve function, comprises the avidity improved CA6.

The antibody improved also comprises those and has the antibody improving feature, and it is formed by animal immune, hybridoma and selects the standard technique with the antibody of special characteristic to be produced.

cytotoxic agent

The cytotoxic agent used in cytotoxic conjugate of the present invention can be any compound, and this compound causes necrocytosis or inducing cell death or reduces cell viability in some way.Such as, preferred cytotoxic agent comprises as undefined CHROMATOGRAPHIC FRACTIONATION AND MASS and CHROMATOGRAPHIC FRACTIONATION AND MASS analogue, Taxane family (taxoids), CC-1065 and CC-1065 analogue, sea hare phallotoxins (dolastatin) and sea hare phallotoxins analogue.The antibody of these cytotoxic agents and antibody disclosed herein, antibody fragment, function equivalent, improvement and their analogue coupling.

Cytotoxic conjugate can be prepared by vitro method.In order to connect medicine or prodrug to antibody, use linking group.The linking group be applicable to is known in this area, comprises disulfide group, thioether group, acid-unstable group, photo-labile group, the unstable group of peptase and the unstable group of esterase.Preferred linking group is disulfide group and thioether group.Such as, two sulphur permutoid reactions can be used or pass through between antibody and medicine or prodrug, form thioether bond and build conjugate.

cHROMATOGRAPHIC FRACTIONATION AND MASS

CHROMATOGRAPHIC FRACTIONATION AND MASS and CHROMATOGRAPHIC FRACTIONATION AND MASS analogue belong to and can be used in the present invention to form the cytotoxic agent of cytotoxic conjugate.The example of the CHROMATOGRAPHIC FRACTIONATION AND MASS be applicable to comprises maytansinol (maytansinol) and maytansinol analogue.CHROMATOGRAPHIC FRACTIONATION AND MASS suppresses microtubule formed and have highly toxic medicine to mammalian cell.

The example of maytansinol analogue be applicable to comprises those aromatic nucleus with modification and those have the medicine of modification in other position.The CHROMATOGRAPHIC FRACTIONATION AND MASS be applicable to like this is disclosed in United States Patent (USP) 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,33I, 598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499 and 5,846, in 545.

The specific examples with the suitable maytansinol analogue of the aromatic nucleus of modification comprises:

(1) C-19-dechlorination (United States Patent (USP) 4,256,746) (also originally being prepared by the LAH of ansamytocin P2);

(2) C-20-hydroxyl (or C-20-demethylation) +/-C-19-dechlorination (United States Patent (USP) 4,361,650 and 4,307,016) (prepared by usage chain mould (Streptomyces) or actinomycetes (Actinomyces) demethylation or the dechlorination using LAH); With

(3) C-20-demethoxylation, C-20-acyloxy (-OCOR), +/-dechlorination (United States Patent (USP) 4,294,757) (preparing by using the acylations of acyl chlorides).

The specific examples with the suitable maytansinol analogue of the modification of other position comprises:

(1) C-9-SH (United States Patent (USP) 4,424,219) is (by maytansinol and H ₂s or P ₂s ₅reaction prepare);

(2) C-14-alkoxyl-methyl (demethoxylation/CH ₂oR) (United States Patent (USP) 4,331,598);

(3) C-14-methylol or acyloxymethyl (CH ₂oH or CH ₂oAc) (United States Patent (USP) 4,450,254) (being prepared by Nocardia (Nocardia));

(4) C-15-hydroxyl/acyloxy (United States Patent (USP) 4,364,866) (by the be converted preparation of streptomycete by maytansinol);

(5) C-15-methoxyl group (United States Patent (USP) 4,313,946 and 4,315,929) (being separated from trewianudiflora (Trewianudiflora));

(6) C-18-N-demethylation (United States Patent (USP) 4,362,663 and 4,322,348) (being prepared by the demethylation of streptomycete by maytansinol); With

(7) 4,5-deoxidations (United States Patent (USP) 4,371,533) (also originally being prepared by the titanous chloride/LAH of maytansinol).

In preferred embodiments, cytotoxic conjugate of the present invention utilizes sulfur-bearing alcohol CHROMATOGRAPHIC FRACTIONATION AND MASS (DM1) to make cytotoxic agent, and it is called N normally ^{2 '}-Tuo acetyl-N ^{2 '}-(3-sulfydryl-1-oxopropyl)-maytenin.DM1 is represented by structural formula (I) below:

In another preferred embodiment, cytotoxic conjugate of the present invention utilizes sulfur-bearing alcohol CHROMATOGRAPHIC FRACTIONATION AND MASS N ^{2 '}-Tuo acetyl-N ^{2 '}-(4-methyl-4-sulfydryl-1-oxopentyl)-maytenin is as cytotoxic agent.DM4 by below structural formula (ID represents:

In the further embodiment of the present invention, can use other maytenin, comprise the CHROMATOGRAPHIC FRACTIONATION AND MASS containing mercaptan and two sulphur things, it is replacing with the carbon atom of sulphur atom having monoalkyl or dialkyl group.These are included in C-3, C-14 methylol; C-15 hydroxyl or C-20 demethylation has the Mei Dengsuo class of the amino acid side chain of acidylate; the amino acid side chain of described acidylate has the acyl group with the sulfhedryl be obstructed; carbon atom wherein with the carboxyl groups of thiol functionalities has one or two substituting group, and described substituting group is CH ₃, C ₂h ₅, there is the linear of 1 to 10 carbon atoms or branched-chain alkyl or alkenyl, the cycloalkyl with 3 to 10 carbon atoms or alkenyl, phenyl, substituted-phenyl or heteroaromatic group or heterocyclic radical; further; one of wherein said substituting group can be H, and wherein said carboxyl groups has the linear chain length of at least three carbon atoms between carbonyl functional group and sulphur atom.

Other maytenin like this comprises the compound represented by formula (III):

Wherein:

Y ' representative

(CR ₇R ₈) _l(CR ₉＝CR ₁₀) _p(C≡C) _qA _o(CR ₅R ₆) _mD _u(CR ₁₁＝CR ₁₂) _r(C≡C) _sB _t(CR ₃R ₄) _nCR ₁R ₂SZ，

Wherein:

R ₁and R ₂be CH independently of one another ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, there is the side chain of 3 to 10 carbon atoms or the alkyl or alkenyl of ring-type, phenyl, substituted-phenyl or heterocyclic aromatic base or heterocyclic radical, in addition, R ₂can be H;

A, B, D have the cycloalkyl of 3-10 carbon atom or cycloalkenyl group, the simple or aryl that replaces or heterocyclic aryl or heterocyclyl groups;

R ₃, R ₄, R ₅, R ₆, R ₇, R ₈, R ₉, R ₁₀, R ₁₁and R ₁₂be H, CH independently of one another ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, there is the side chain of 3 to 10 carbon atoms or the alkyl or alkenyl of ring-type, phenyl, substituted-phenyl or heterocyclic aryl or heterocyclyl groups;

L, m, n, o, p, q, r, s, t and u be independently of one another 0 or from 1 to 5 integer, condition be l, m, n, o, p, q, r, s, t and u at least two different time be 0; With

Z is H, SR or-COR, and wherein R is linear alkyl or the thiazolinyl with 1 to 10 carbon atoms, has branch or ring-type the alkyl or alkenyl of 3 to 10 carbon atoms, or the simple or aryl that replaces or heterocyclic aryl or heterocyclyl groups.

The preferred embodiment of formula (III) comprises the compound of such formula (III),

Wherein:

R ₁methyl, R ₂be H, Z be H;

R ₁and R ₂be methyl, Z is H;

R ₁methyl, R ₂be H, Z be-SCH ₃;

R ₁and R ₂be methyl, Z is-SCH ₃.

Other maytenin so also comprises the compound represented by formula (IV-L), (IV-D) or (IV-D, L):

Wherein:

Y represents (CR ₇r ₈) _l(CR ₅r ₆) _m(CR ₃r ₄) _ncR ₁r ₂sZ,

Wherein:

R ₁and R ₂be CH independently of one another ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclic radical, in addition, R ₂can be H;

R ₃, R ₄, R ₅, R ₆, R ₇and R ₈respective is independently H, CH ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclyl groups;

L, m and n are the integer of 1 to 5 independently of one another, and in addition, n can be 0;

Z is H, SR or-COR, and wherein R has the alkyl or alkenyl of the linear of 1 to 10 carbon atoms or branch, the cycloalkyl with 3 to 10 carbon atoms or cycloalkenyl group, or the simple or aryl that replaces or heterocyclic aryl or heterocyclyl groups; With

May represents CHROMATOGRAPHIC FRACTIONATION AND MASS (maytansinoid), its at C-3, C-14 methylol, C-15 hydroxyl or C-20 demethyl place with side chain.

The preferred embodiment of formula (IV-L), (IV-D) and (IV-D, L) comprises such formula (IV-L), the compound of (IV-D) and (IV-D, L), wherein:

R ₁for methyl, R ₂h, R ₅, R ₆, R ₇and R ₈each be H, l and m each be 1, n be 0, and Z is H.

R ₁and R ₂for methyl, R ₅, R ₆, R ₇, R ₈each be H, l and m be 1, n is 0, and Z is H.

R ₁for methyl, R ₂h, R ₅, R ₆, R ₇and R ₈each be each 1, the n of being of H, l and m is 0, and Z is-SCH ₃.

R ₁and R ₂for methyl, R ₅, R ₆, R ₇, R ₈each be H, l and m be 1, n is 0, and Z is-SCH ₃.

Preferred cytotoxic agent is represented by formula (IV-L).

Other maytenin so also comprises by the compound shown in formula (V):

Wherein:

Y represents (CR ₇r ₈) _l(CR ₅r ₆) _m(CR ₃r ₄) _ncR ₁r ₂sZ,

Wherein:

R ₁and R ₂be CH independently of one another ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclyl groups, in addition, R ₂can be H;

R ₃, R ₄, R ₅, R ₆, R ₇and R ₈be H, CH independently of one another ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclyl groups;

L, m and n are the integer of 1 to 5 independently of one another, and in addition, n can be 0; With

Z is H, SR or-COR, and wherein R has the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, or the simple or aryl that replaces or heterocyclic aryl or heterocyclyl groups.

The preferred embodiment of formula (V) comprises the compound of such formula (V), wherein:

R ₁for methyl, R ₂h, R ₅, R ₆, R ₇and R ₈each be each 1, the n of being of H, l and m is 0, and Z is H.

Other maytenin like this comprises by the compound shown in formula (VI-L), (VI-D) or (VI-D, L) further:

Wherein:

Y ₂representative (CR ₇r ₈) _l(CR ₅r ₆) _m(CR ₃r ₄) _ncR ₁r ₂sZ ₂,

Wherein:

R ₃, R ₄, R ₅, R ₆, R ₇and R ₈be H, CH independently of one another ₃, C ₂h ₅, there is the linear loop alkyl or alkenyl of 1 to 10 carbon atoms, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclyl groups;

Z ₂for SR or COR, wherein R is branch or ring-type the alkyl or alkenyl having the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, have 3 to 10 carbon atoms, or the simple or aryl that replaces or heterocyclic aryl or heterocyclyl groups; With

May is CHROMATOGRAPHIC FRACTIONATION AND MASS.

Other maytenin so also comprises the compound represented by formula (VII):

Wherein:

Y ₂' representative

(CR ₇r ₈) _l(CR ₉=CR ₁₀) _p(C ≡ C) _qa _o(CR ₅r ₆) _md _u(CR ₁₁=CR ₁₂) _r(C ≡ C) _sb _t(CR ₃r ₄) _ncR ₁r ₂sZ ₂, wherein:

R ₁and R ₂be CH independently of one another ₃, C ₂h ₅, there is the linear of 1 to 10 carbon atoms or branched alkyl groups or thiazolinyl, the cycloalkyl with 3 to 10 carbon atoms or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclic radical, in addition, R ₂can be H;

A, B and D are independently of one another for having the cycloalkyl of 3 to 10 carbon atoms or cycloalkenyl group, the simple or aryl that replaces or heterocyclic aryl or heterocycloalkyl;

R ₃, R ₄, R ₅, R ₆, R ₇, R ₈, R ₉, R ₁₀, R ₁₁and R ₁₂each is H, CH independently ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclyl groups;

L, m, n, o, p, q, r, s, t and u are each be independently 0 or from 1 to 5 integer, condition be l, m, n, o, p, q, r, s, t and u at least two different time be zero; With

Z ₂for SR or-COR, wherein R be have 1 to 10 carbon atoms linear alkyl or thiazolinyl, there is the branch of 3 to 10 carbon atoms or the alkyl or alkenyl of ring-type, or the simple or aryl that replaces or heterocyclic aryl or heterocyclyl groups.

The preferred embodiment of formula (VII) comprises the compound of such formula (VII), wherein: R ₁for methyl and R ₂for H.

Above-mentioned CHROMATOGRAPHIC FRACTIONATION AND MASS can be coupled to anti-CA6 antibody DS6 or its homologue or its fragment; wherein use mercaptan or two sulphur functional groups that antibody is connected to CHROMATOGRAPHIC FRACTIONATION AND MASS; described mercaptan or two sulphur functional groups be present in C-3, C-14 methylol of CHROMATOGRAPHIC FRACTIONATION AND MASS, C-15 hydroxyl or C-20 demethylation place acylated amino side chain carboxyl groups on; wherein the mercaptan that has of the carboxyl groups of the amino acid side chain of acidylate or two sulphur functional groups are positioned at and have on one or two substituent carbon atom, and described substituting group is CH ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclyl groups; in addition; one of substituting group can be H, and wherein said carboxyl groups has the linear chain length of at least three carbon atoms between carbonyl functional group and sulphur atom.

The preferred conjugate of the present invention is for comprising the conjugate of anti-CA6 antibody DS6 with the coupling of formula (VIII) CHROMATOGRAPHIC FRACTIONATION AND MASS or its homologue or its fragment:

Wherein:

Y ₁' representative

(CR ₇R ₈) _l(CR ₉＝R ₁₀) _p(C≡C) _qA _o(CR ₅R ₆) _mD _u(CR ₁₁＝CR ₁₂) _r(C≡C) _sB _t(CR ₃R ₄) _nCR ₁R ₂S-，

Wherein:

A, B and D are separately independently for having the cycloalkyl of 3 to 10 carbon atoms or cycloalkenyl group, the simple or aryl that replaces, or heterocyclic aryl or heterocyclyl groups;

R ₃, R ₄, R ₅, R ₆, R ₇, R ₈, R ₉, R ₁₀, R ₁₁and R ₁₂each is H, CH independently ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the side chain with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclyl groups; With

L, m, n, o, p, q, r, s, t and u are each be independently 0 or 1 to 5 integer, condition is l, m, n, o, p, q, r, s, t and u at least two is not be zero simultaneously.

Preferably, R ₁methyl is and R ₂for H or R ₁and R ₂for methyl.

The preferred conjugate of the present invention is for comprising and formula (IX-L), (IX-D) or the anti-CA6 antibody DS6 of (IX-D, L) CHROMATOGRAPHIC FRACTIONATION AND MASS coupling or the conjugate of its homologue or fragment:

Wherein:

Y ₁representative (CR ₇r ₈) _l(CR ₅r ₆) _m(CR ₃r ₄) _ncR ₁r ₂s-,

Wherein:

R ₃, R ₄, R ₅, R ₆, R ₇and R ₈each is H, CH independently ₃, C ₂h ₅, there is the linear alkyl of 1 to 10 carbon atoms or thiazolinyl, the branch with 3 to 10 carbon atoms or cyclic alkyl or thiazolinyl, phenyl, the phenyl of replacement or heterocyclic aryl or heterocyclyl groups;

L, m and n are each be independently 1 to 5 integer, in addition, n can be 0; With

May represents maytansinol, and it has and is positioned at C-3, C-14 methylol, C-15 hydroxyl or C-20 demethyl side chain everywhere.

The preferred embodiment of formula (IX-L), (IX-D) and (IX-D, L) comprises such formula (IX-L), the compound of (IX-D) and (IX-D, L), wherein:

R ₁for methyl and R ₂for H, or R ₁and R ₂for methyl,

R ₁for methyl, R ₂for H, R ₅, R ₆, R ₇and R ₈each be each 1, the n of being of H, l and m is 0,

R ₁and R ₂for methyl, R ₅, R ₆, R ₇and R ₈each be H, l and m be 1, n is 0.

Preferably, cytotoxic agent is represented by formula (IX-L).

The present invention further preferred conjugate for comprising anti-CA6 antibody DS6 or its homologue or fragment and the conjugate of CHROMATOGRAPHIC FRACTIONATION AND MASS coupling with formula (X):

Wherein, substituting group as in above-mentioned formula (IX) define.

Particularly preferably be any above-claimed cpd, wherein R ₁for H, R ₂for methyl, R ₅, R ₆, R ₇and R ₈each be H, l and m each be 1, and n is 0.

Particularly preferred is further any above-claimed cpd, wherein R ₁and R ₂for methyl, R ₅, R ₆, R ₇, R ₈each be H, l and m is 1, and n is 0.

Further, L-aminoacyl steric isomer is preferred.

In the U.S. Patent application 10/849 just on the docket that May 20 in 2004 submits to, the various CHROMATOGRAPHIC FRACTIONATION AND MASS described in 136 also can be used in cytotoxic conjugate of the present invention.Be incorporated herein U.S. Patent application 10/849, whole disclosures of 136 as a reference.

containing the linking group of disulphide

In order to CHROMATOGRAPHIC FRACTIONATION AND MASS be connected with Cell binding agent such as DS6 antibody, CHROMATOGRAPHIC FRACTIONATION AND MASS comprises connection portion.This connection portion comprises chemical bond, and this chemical bond allows completely active CHROMATOGRAPHIC FRACTIONATION AND MASS to discharge at privileged site.The chemical bond be applicable to is known in this area, comprises disulfide linkage, acid labile key, photo-labile key, peptase unstable key and esterase unstable key.Preferred disulfide linkage.

Connection portion also comprises active chemical group.In a preferred embodiment, described active chemical group can by disulfide linkage connection portion and CHROMATOGRAPHIC FRACTIONATION AND MASS covalently bound.

Particularly preferred active chemical group is N-succinimide ester (N-succinimidyl ester) and N-sulfosuccinimide ester (N-sulfosuccinimidyl esters).

The particularly preferred CHROMATOGRAPHIC FRACTIONATION AND MASS comprising connection portion containing active chemical group is C-3 ester and the analogue thereof of maytansinol, and wherein said connection portion comprises disulfide linkage, and active chemical group comprises N-succinimide ester or N-sulfosuccinimide ester.

A lot of positions in CHROMATOGRAPHIC FRACTIONATION AND MASS can connect the position of described connection portion as chemistry.Such as, the C-14 position there is the C-3 position of hydroxyl, modifying with methylol, be all considered to useful with the C-15 position of hydroxyl modified and the C-20 position with hydroxyl.But, preferred C-3 position, and the C-3 position of particularly preferably maytansinol.

Although according to the connection portion containing disulfide linkage, describe the synthesis of the ester of the maytansinol with connection portion, but those skilled in the art will appreciate that, the connection portion with other chemical bond (as mentioned above) also can be used to the present invention, can be used to the present invention as other CHROMATOGRAPHIC FRACTIONATION AND MASS.The object lesson of other chemical bond comprises sour labile bond, photo-labile key, peptase labile bond and esterase labile bond.At the United States Patent (USP) 5,208 that this introduces, the disclosure of 020 teaches the preparation of the CHROMATOGRAPHIC FRACTIONATION AND MASS with such key.

At United States Patent (USP) 6,441,163 and 6,333,410 and U. S. application number 10/161, describe the synthesis of CHROMATOGRAPHIC FRACTIONATION AND MASS and the CHROMATOGRAPHIC FRACTIONATION AND MASS derivative had containing the disulfide moieties of active group in 651, every section of patent is incorporated herein by reference.

By the CHROMATOGRAPHIC FRACTIONATION AND MASS such as DM1 containing active group, with antibody such as DS6 antibody response, to produce cytotoxic conjugate.These conjugates of purifying can be carried out by HPLC or gel-filtration.

United States Patent (USP) 6,333,410 and U. S. application numbers 09/867,598,10/161,651 and 10/024, provide the several good scheme for generation of such antibody-CHROMATOGRAPHIC FRACTIONATION AND MASS conjugate in 290, every section of patent is incorporated herein with its full content.

Generally speaking, can by the CHROMATOGRAPHIC FRACTIONATION AND MASS of molar excess of disulfide moieties that has containing active group and the antibody-solutions incubation in water-containing buffering liquid.By adding excess amine (such as thanomin, taurine etc.) cancellation reaction mixture.Then purifying CHROMATOGRAPHIC FRACTIONATION AND MASS-antibody coupling matter is carried out by gel-filtration.

By the absorbance ratio at spectrophotometry 252nm and 280nm place, the quantity of the CHROMATOGRAPHIC FRACTIONATION AND MASS molecule that each antibody molecule combines can be measured.Preferably average 1-10 CHROMATOGRAPHIC FRACTIONATION AND MASS molecule/antibody molecule.

The conjugate can evaluating antibody and CHROMATOGRAPHIC FRACTIONATION AND MASS medicine suppresses the ability of various less desirable cell line proliferation in vitro.Such as, clone such as people's epidermal carcinoma cell lines A-431, human small cell lung carcinoma clone SW2, human breast tumor cell line SKBR3 and Burkitt lymphoma cell line Namalwa can easily be used to the cytotoxicity evaluating these compounds.Cell to be evaluated can be exposed to compound 24 hours, in directly measuring, be measured the surviving fraction of cell by known method.Then IC can be calculated by measurement result ₅₀value.

containing the linking group of PEG

Also can use PEG linking group that CHROMATOGRAPHIC FRACTIONATION AND MASS is connected to Cell binding agent, as at U. S. application number 10/024, described in 290.These PEG linking groups are all solvable in water and non-aqueous solvent, and one or more cytotoxic agent can be used to be connected to Cell binding agent.Exemplary PEG linking group comprises different bifunctional PEG joint, and cytotoxic agent and Cell binding agent are connected to the two ends of joint by it by the merit functionality sulfydryl of one end or the active ester of disulfide group and the other end.

As the general example using PEG linking group synthetic cell cytotoxic conjugate, its detail also can with reference to U. S. application numbers 10/024,290.Synthesis starts from one or more cytotoxic agent of making to have active peg moiety and Cell binding agent is reacted, cause the end active ester of each active peg moiety by the radical amino acid replacement of Cell binding agent, comprise the cytotoxic conjugate of one or more cytotoxic agent to produce, described cytotoxic agent through PEG linking group and Cell binding agent covalently bound.

taxanes

The cytotoxic agent be used in cytotoxic conjugate of the present invention also can be Taxan or derivatives thereof.

Taxan (taxane) class is a compounds family, it comprises taxol (paclitaxel) (Taxol), it is cytotoxicity natural product, with Docetaxel (docetaxel) (Taxotere), it is semi-synthetic derivative, and these two kinds of compounds are widely used in the treatment of cancer.Taxanes is mitotic spindle toxic agent, and it suppresses the depolymerization of tubulin, thus causes necrocytosis.Although in the treatment of cancer, Docetaxel and taxol are useful medicaments, but their anti-tumor activity is limited, reason is that they have non-specific toxicity to normal cell.In addition, the compound as taxol and Docetaxel itself can not be applied in enough effectively with the conjugate of Cell binding agent.

Preferred Taxan for the preparation of cytotoxic conjugate is the Taxan of formula (XI):

The synthetic method of the taxanes in cytotoxic conjugate of the present invention can be used in, and the coupling method of taxanes and cytotoxic agent such as antibody, at United States Patent (USP) 5,416,064,5,475,092,6,340,701,6,372,738 and 6,436,931 and at U. S. application number 10/024,290,10/144,042,10/207,814,10/210,112 and 10/369, be described in detail in 563.

cC-1065 analogue

Being applied according to the cytotoxic agent in cytotoxic conjugate of the present invention also can be CC-1065 or derivatives thereof.

CC-1065 is the effective antitumour microbiotic separated from the culture of damp ear streptomycete (Streptomyces zelensis).CC-1065 is in vitro than the drug effect height about 1000 times of the anticarcinogen of common use, anticarcinogen such as Zorubicin (doxorubicin), methotrexate (methotrexate) and vincristine(VCR) (the vincristine) (B.K.Bhuyan etc. of common use, Cancer Res., 42,3532-3537 (1982)).CC-1065 and analogue thereof at United States Patent (USP) 6,372,738,6,340,701,5,846,545 and 5,585, be disclosed in 499.

The cytotoxic effects of CC-1065 combines to its alkylation activity and its DNA or DNA embeds active relevant.These two kinds of activity are positioned at the different piece of this molecule.Therefore, alkylation activity is included in ring third pyrrolo-indole (cyclopropapyrroloindole) (CPI) subunit, and DNA binding activity is arranged in two pyrrolo-indole (pyrroloindole) subunits.

Although CC-1065 has certain attracting feature as cytotoxic agent, but it has limitation on therepic use.Use CC-1065 to mouse and can cause retardance liver toxicity, this causes the 50th day dead { V.L.Reynolds etc., J.Antibiotics, XXIX, 319-334 (1986) } after single dose intravenous dosage 12.5 μ g/kg.This has encouraged exploitation not cause the effort of the analogue of delayed toxicity, is that the synthesis of the simpler analogue of model is described { M.A.Warpehoski etc., J.Med, Chem., 31,590-603 (1988) } with CC-1065.

In another a series of analogue, CPI part is replaced { D.L.Boger etc. by ring propyl benzene diindyl (cyclopropabenzindole) (CBI) part, J.Org.Chem., 55,5823-5833, (1990), D.L.Boger etc., BioOrg.Med.Chem.Lett., 1,115-120 (1991) }.These compounds maintain the high vitro efficacy of parent drug, in mouse, do not cause delayed toxicity.Similar CC-1065, these compounds are alkylating agents, its with covalent manner in conjunction with the ditch of DNA to cause necrocytosis.But, disappointed result { B.F.Foster etc. have been produced to the analogue U 73975 (Adozelesin) of most prospect and the clinical assessment of U 80244 (Carzelesin), Investigational New Drugs, 13,321-326 (1996); I.Wolff etc., Clin.CancerRes., 2,1717-1723 (1996) }.These medicines show not good result for the treatment of, and reason is that it has high general toxicity.

Change distribution in vivo by directional transmissions to tumor locus, greatly can improve the therapeutic efficiency of CC-1065 analogue, thus make, to non-target tissue, there is low toxicity, and therefore there is lower general toxicity.For realizing this target, the conjugate of the Cell binding agent of the analogue of CC-1065 and derivative and selectively targeted tumour cell is described { United States Patent (USP): 5,475,092; 5,585,499; 5,846,545}.These conjugates typically demonstrate high target-specific cytotoxin in vitro, and demonstrate outstanding anti-tumor activity { R.V.J.Chari etc. in people's tumor xenograft models in mouse, Cancer Res., 55,4079-4084 (1995) }.

Can be used on the synthetic method of the CC-1065 analogue in cytotoxic conjugate of the present invention, and the coupling method of described analogue and Cell binding agent such as antibody is at United States Patent (USP) 5,475,092,5,846,545,5,585,499,6,534,660 and 6,586,618 and at U. S. application 10/116,053 and 10/265, be described in detail in 452.

other medicines

Medicine is methotrexate (methotrexate) such as, daunorubicin (daunorubicin), Zorubicin (doxorubicin), vincristine(VCR) (vincristine), vinealeucoblastine(VLB) (vinblastine), melphalan (melphalan), ametycin (mitomycin C), Chlorambucil (chlorambucil), calicheamicin (calicheamicin), tubulysin and tubulysin analogue, duocarmycin and duocarmycin analogue, sea hare phallotoxins and sea hare phallotoxins analogue are also applicable to preparation conjugate of the present invention.Described drug molecule can be connected with antibody molecule by intermediary carrier molecule such as serum albumin.Such as, Doxarubicin and Danorubicin compound also can be useful cytotoxic reagent, as described in United States serial 09/740991.

therapeutic composition

Present invention provides therapeutic composition, it comprises:

One or more cytotoxic conjugates of (a) significant quantity; With

(b) pharmacology acceptable carrier.

Similarly, the invention provides the method for the growth for suppressing the cell mass selected, the method comprises: with the cytotoxic conjugate of significant quantity or comprise the therapeutic agent target cell of cytotoxic conjugate or comprise the tissue of target cell, and described cytotoxic conjugate or therapeutical agent are used separately or used with other cytotoxic agent or therapeutic agent.

The present invention also comprises the method using therapeutic composition of the present invention treatment cancer stricken object.

By previously described method, cytotoxic conjugate tiring and specificity (such as, see, R.V.J.Chari etc., Cancer Res.55:4079-4084 (1995)) in vitro can be evaluated.By the method previously also described, anti-tumor activity (see such as Liu etc., Proc.Natl.Acad.Sci.93:8618-8623 (1996)) can be evaluated in people's tumor xenograft models of mouse.

Suitable pharmacology acceptable carrier is known, and those of ordinary skill in the art can determine whether it meets clinical setting license.As utilized herein, carrier comprises thinner and vehicle.

The example of suitable carrier, thinner and/or vehicle comprises: (1) Dulbecco phosphate buffered saline(PBS), pH ~ 7.4, comprise or do not comprise about 1mg/ml to 25mg/ml human serum albumin, (2) 0.9% salt solution (0.9%w/v sodium-chlor (NaCl)), and (3) 5% (w/v) glucose; Also antioxidant such as tryptamines and stablizer such as Tween 20 can be comprised.

The method of the growth of the cell mass selected by suppression can (in vitro), in vivo (in vivo) or in vitro (ex vivo) be implemented in vitro.As utilized herein, Developing restraint mean the cell that slows down growth, reduce cell survival rate, cause the death of cell, lysing cell and inducing cell death, no matter be in short time durations or long time durations.

The example of external use is included in the process before to autologous bone marrow in autologous bone marrow transplantation to same patient body, and object kills ill or pernicious cell; Process before bone marrow transplantation, object kills competitive T cell and prevention graft versus host disease (GVH disease) (graft-versus-host-disease (GVHD)); The process of cell culture, object kills all cells except the expectation variant of not expressing target antigen; Or kill the variant of expressing less desirable antigen.

Those of ordinary skill in the art easily can determine the situation of the external application of non-clinical.

The example of in vitro clinical application is in cancer therapy or treating autoimmune diseases, before autotransplantation, tumour cell or lymphocyte is removed from marrow, or before transplanting, from autologous or allogenic bone marrow or tissue, remove T cell and other lymphocyte, object is prevention graft versus host disease (GVH disease) (graft versus hostdisease (GVHD)).Treatment can be carried out as follows.Individual gather marrow from patient or other, then add incubation in the substratum of cytotoxic agent of the present invention containing serum.Concentration about 10 μMs to 1pM, about 37 DEG C of incubations about 30 minutes to about 48 hours.Those of ordinary skill in the art can easily determine definite concentration conditions and incubative time, i.e. dosage.After incubation, with the substratum washing medullary cell containing serum, and be back to patient by venoclysis in accordance with known methods.In some cases, gathering the period between marrow and the cell that infusion is processed again, patient accepts other treatment and such as melts chemotherapy or Systemic radiation process, now, uses the medical facilities of standard by processed medullary cell freezen protective in liquid nitrogen.

For clinical application in vivo, cytotoxic conjugate of the present invention will be supplied in the form of a solution, test to the sterility of this solution and level of endotoxin.The example of the suitable application program of cytotoxic conjugate is as follows.Conjugate is given weekly, totally 4 weeks in the mode of vein pill (bolus) administration weekly.Pellet dosages is provided in the physiological saline of 50 to 100ml, wherein can add the human serum albumin of 5 to 10ml.Dosage is each administration 10 μ g to 100mg, the intravenously administrable scope of 100ng to 1mg/kg (every day).More preferably, dosage range is 50 μ g to 30mg.Most preferably, dosage range is 1mg to 20mg.After treatment surrounding, patient can continue to accept treatment based on week.When clinical setting is permitted, those of ordinary skill in the art can determine the concrete clinical protocol such as relevant route of administration, vehicle, thinner, dosage, number of times.

According to kill selection cell colony body in or ex vivo approach, the example of the medical condition that can be treated comprises the malignant diseases of any type, comprise sarcoma or cancer that such as lung cancer, breast cancer, colorectal carcinoma, prostate cancer, kidney, carcinoma of the pancreas, ovarian cancer, cervical cancer and lymphoid organ cancer, osteosarcoma, synovial membrane cancer, wherein CA6 be expressed and wherein CA6 sugar epi-position by other cancer to be determined obviously expressed; Autoimmune disease, such as systemic lupus erythematous, rheumatoid arthritis and multiple sclerosis; Transplant rejection, such as renal transplant rejection, liver transplantation repulsion, lung transplant rejection, cardiac transplant rejection episode and marrow graft rejection; Graft versus host disease (GVH disease); Virus infection, such as mV infection, HIV, AIDS etc.; And parasitic infection, the Other diseases that such as giardiasis, loeschiasis, schistosomicide and those of ordinary skill in the art determine.

test kit

The present invention also comprises test kit, and it such as comprises described cytotoxic conjugate and uses this cytotoxic conjugate to kill the operation instruction of particular cell types.Described operation instruction can comprise the guidance for the cytotoxic conjugate of use in vitro, in vivo or in vitro.

Typically, test kit will have the compartment (compartment) comprising cytotoxic conjugate.Cytotoxic conjugate can be other form that lyophilized form, liquid form maybe can be suitable for being comprised in test kit.Test kit also can comprise for the other component needed for the method that the specification sheets implemented in test kit describes, such as reconstructing the sterile solution of lyophilized powder, in the additional agent combined with cytotoxic conjugate before patient's administration, and help the instrument using conjugate to patient.

other embodiment

The present invention further provides monoclonal antibody, humanized antibody and epitope binding fragments thereof, they are marked further for use in research or diagnostic use.In preferred embodiments, radio-labeling, fluorophore, chromophore, developer or metal ion is labeled as described in.

Also provide diagnostic method, the antibody of wherein said mark or its epitope binding fragments are applied to the cancered object of suspection, and measure or be marked at the distribution in subject described in monitoring.

Embodiment

Can understand scope widely of the present invention best with reference to the following example, these embodiments are also not intended to make the present invention be limited to specific embodiment.

embodiment 1: the qualification of the antigen positive undertaken by flow cytometry binding tests and negative cells system

Flow cytometry is used to DS6 epi-position, CA6 to navigate to cell surface.Human cell line is from American type culture collection (American Type Culturae Collection, ATCC) obtain, except OVCAR5 (Kearse etc., Int.J.Cancer 88 (6): 866-872 (2000)), outside OVCAR8 and IGROV1 cell (M.Seiden, Massachusetts General Hospital).All cells are cultivated in RPMI 1640, wherein be supplemented with 4mM L-glutaminate, 50U/ml penicillin, 50 μ g/ml Streptomycin sulphate (CambrexBio Science, Rockland, and 10%v/v foetal calf serum (Atlas Biologicals ME), Fort Collins, CO), it is hereafter being referred to as substratum.Cell is maintained at 37 DEG C, 5%CO ₂in moistening incubation case.

By cell (1-2 × 10 ⁵individual cells/well) with the DS6 antibody of serial dilution concentration at incubated on ice 3-4h, described DS6 antibody in 96 orifice plates, preparation in FACS damping fluid (2% sheep blood serum, RPMI).At 4 DEG C, with 1500rpm spun down cell 5 minutes in desk centrifuge.After removing substratum, then refill hole with the FACS damping fluid of 150 μ l.Then repeated washing step.Sheep anti-mouse igg (Jackson hnmunoresearch) the FACS damping fluid dilution 1: 100 of FITC-mark, and with cell at incubated on ice 1h (hour).Tissue Culture Plate is covered, in case the photobleaching of stop signal with paper tinsel.After twice washing, with 1% formaldehyde fixed cell, and analyze on flow cytometer.

Significantly, CA6 epi-position is found in the clone (table 3) in ovary, mammary gland, uterine cervix and pancreas source, and as tumour immunity, histological chemistry predicted.But some clones of other tumor type demonstrate limited CA6 and express.DS6 antibodies has the apparent K of 135.6pM _d(in PC-3 cell, table 3).In antigen-positive cell system, the maximum mean fluorecence (table 3) of binding curve (Fig. 1) illustrates relative antigen density.

Table 3

* average maximum relative mean fluorescent

the sign of embodiment 2:DS6 epi-position

By carrying out immunoblotting to the Dot blot of CA6 positive cell split product (Caov-3), analyze the character of DS6 antigens c A6, described product of cell lysis proteolysis (PRONASE A and Proteinase K) and/or sugared cracking (neuraminidase and periodic acid) process digest.For positive control, on the lysate of antigen-positive cell system, test identifies other antibody (Caov-3 and CM1 of various epi-position type; Colo205 and C242; SKMEL28 and R24).CM1 is the antibody of the protein epitope of variable number series winding repeating structure territory (variablenumber tandem repeat domain (VNTR)) identifying Muc-1, therefore for protein epitope provides contrast.C242 is combined in new colorectal carcinoma specificity sialic acid dependency sugar epi-position (CanAg) on Muc-1, and it is that sugared epi-position on albumen provides contrast.R24 combines and is specific to melanomatous GD3 Sphingolipids,sialo, therefore for the sugared epi-position on nonprotein skeleton provides contrast.

Caov-3, Colo205 and SKMEL28 cell is laid in 15cm tissue culturing plate.In the day before yesterday of cracking, upgrade substratum (30mL/ plate).At RIPA damping fluid (50mM Tris-HClpH 7.6,150mM NaCl, the 5mM EDTA of the improvement of precooling on ice, 1%NP40,0.25% sodium deoxycholate), proteinase inhibitor (PMSF, pepstatin A, leupeptin and AKOLINE) and PBS.Substratum after sucking-off, is washed cell twice with the cold PBS of 10ml from plate.All steps are subsequently carried out on ice and/or in 4 DEG C of cold houses.After the last washes of PBS is sucked out, with 1-2mL lysis buffer (RIPA damping fluid, with the fresh proteinase inhibitor added, ultimate density is 1mM PMSF, 1 μM of pepstatin A, 10 μ g/ml are bright presses down proteolytic enzyme and 2 μ g/ml AKOLINEs) lysing cell.Use cell harvestor (cell lifter) to scrape split product from flat board, and use 18G pin to be pulverized by split product by upper and lower pressure-vaccum suspension (5-10 time).By split product vortex 10 minutes, then in micro-whizzer with centrifugal 10 minutes of maximum speed of revolution (13K rpm).Remove precipitation, then use Bradford protein assay kit (Biorad) cls analysis supernatant liquor.

Split product (2 μ l) is directly pipetted on the nitrocellulose membrane of 0.2 μm of drying.Make spot air-dry about 30 minutes.This film is cut into pieces, and every sheet comprises a point.At 37 DEG C, there is PRONASE A (1mg/ml enzyme, 50mM Tris pH 7.5,5mM CaCl ₂), Proteinase K (1mg/ml enzyme, 50mM TrispH 7.5,5mM CaCl ₂), neuraminidase (20mU/ml enzyme, 50mM sodium-acetate pH 5,5mM CaCl ₂, 100 μ g/ml BSA) or periodic acid (20mM, 0.5M sodium-acetate pH 5) condition under by spot incubation 1h.Reagent is purchased from Roche (enzyme) and VWR (periodic acid).Film is at T-TBS lavation buffer solution (0.1%Tween 20,1 × TBS) middle washing (5 minutes), at room temperature use Block buffer (3%BSA, T-TBS) to close 2h, and 4 DEG C in Block buffer with 2 μ g/ml primary antibodie (i.e. DS6, CM1, C242, R24) Overnight incubation.With T-TBS, film is washed three times, 5 minutes, then at room temperature, with anti-(the Jackson Immunoresearch of sheep anti mouse (or people) IgG bis-of HRP coupling; 1: 2000 diluent in confining liquid) incubation 1h.Immunoblotting is washed three times, use ECL system (Amersham) development.

Digested contrast split product immunoblotting (Fig. 2) display, CM1 signal is destroyed by proteolytic treatment, and the signal of sugared cracking digest is uninfluenced, as identify protein epitope antibody expected.C242 signal is destroyed by proteolysis or sugared cracking process, as to be identified in the sugared epi-position that albumen finds antibody expected.R24 signal is by the impact of proteolytic treatment, and it is destroyed by neuraminidase or periodic acid process, as identify Sphingolipids,sialo antibody expected.The DS6 immunoblotting display of digested Caov-3 split product Dot blot, loses with signal after proteolysis and sugared cracking compound treatment.Therefore, the sugared epi-position in protein core (proteinaceous core) is combined in as C242, DS6.In addition, the signal in DS6 immunoblotting is responsive to neuraminic acid ferment treatment.Therefore, CA6, as CanAg, is sialic acid dependency sugar epi-position.

In order to confirm the sugar essence of CA6, Caov-3 split product is put on pvdf membrane also with the de-glycosylation reagent of chemistry, trifluoromethanesulfonic acid (trifluoromethane sulfonic acid) (TFMSA), in nitrogen atmosphere, processes 5 minutes under room temperature.Wash spot with T-TBS, and carry out immunoblotting (Fig. 3) with CM1 or DS6.DS6 signal is destroyed after with acid treatment, this further offers evidence and shows that CA6 is sugared epi-position.After TFSMA process, CM1 signal strengthens, and shows that this acid treatment does not affect the protein in filtrate, and shows that sugared cracking process exposes the protein epitope identified by CM1.

In order to explain the structure of the sugar holding CA6 further, with N-glycanase, O-glycanase and/or sialidase digestion Dot blot (Fig. 4).At 100 DEG C, with denaturation buffer (Glyko) the incubation Caov-3 product of cell lysis (100 μ gs, 30 μ ls) 5 minute of 2.5 μ l containing SDS and beta-mercaptoethanol.Then at 37 DEG C, with 1 μ l N-glycanase, O-glycanase and/or sialidase A (Glyko) by this denatured lysis product digestion 1h.Then by the split product point of digestion on (2 μ l) soluble cotton, and as above-mentionedly carry out immunoblotting.

N-glycanase has no significant effect DS6 immunoblotting signal.But, do not produce signal with the sample of sialidase digestion.Because O-glycanase can not digest connect sugar without the pretreated sialylated O-of sialidase, so use separately the DS6 signal of the sample of O-glycanase process by unaffected.On the contrary, the activity of N-glycanase does not need to carry out pre-treatment with any Glycosylase.The fact that the process of N-glycanase does not affect DS6 signal shows, CA6 epi-position is very likely positioned at sialylated O-and joins on sugar chain.

embodiment 3: to the explaination of antigen finding that there is CA6 epi-position thereon

In order to identify the antigen finding that there is CA6 sialic acid sugar epi-position thereon, by SDS-PAGE and western blot analysis DS6 immunoprecipitate.At 4 DEG C, cell lysate supernatant (1mL/ sample; 3-5mg protein) with the Protein G pearl balanced by 1ml RIPA damping fluid (30 μ l) pre-cleaning 1-2h, along with rotation.On ice and/or 4 DEG C of cold houses carry out all subsequent steps.Spun down pre-cleaning pearl (2-3s) in short-term in Eppendorf centrifuge.Pretreated supernatant liquor is transferred in new pipe, and is incubated overnight under rotating state with 2 μ g DS6.The Protein G pearl (30 μ l) of freshly prepd balance is added in split product, and under rotating state incubation 1h.Spun down pearl-lysate suspension in short-term in Eppendorf centrifuge, optionally takes out the sample of lysate after immunoprecipitation.With 1mL RIPA damping fluid by bead wash 5-10 time.

Then at 37 DEG C with 30 μ l neuraminidases (20mU neuraminidase (Roche), 50mM sodium acetate pH 5,5mM CaCl ₂, 100 μ g/ml BSA) or 30 μ l periodic acids (20mM periodic acid (VWR), 0.5M sodium acetate pH 5) digestion immunoprecipitation DS6 sample 1h.Then be resuspended in the 2 × sample loading buffer (containing beta-mercaptoethanol) of 30 μ l.Pearl is boiled 5 minutes, and sample loading buffer supernatant liquor is loaded to (Invitrogen) in 4-12% or 4-20%Tris-glycine gels.At 125V, this gel is run glue 1.5h in Laemmli electrophoretic buffer.Use Mini Trans-blot transfer device (Biorad), gel sample is transferred to (Invitrogen) on the nitrocellulose membrane of 0.2 μm, spend the night under 20mA.As described in above-described embodiment 2, with this film of DS6 immunoblotting.

Alternatively, first by the pearl sex change of immunoprecipitation, then use N-glycanase, O-glycanase and/or sialidase A (Glyko) enzymatic digestion.Pearl is resuspended in 27 μ l incubation buffering liquids and 2 μ l sex change liquid (Glyko), and 100 DEG C of incubations 5 minutes.After cool to room temperature, add detergent solution (2 μ l) and with 1 μ l N-glycanase, O-glycanase and/or sialidase A at 37 DEG C by sample incubation 4 hours.After adding 5 × sample loading buffer (7 μ l), sample is boiled 5 minutes.As mentioned above, sample is made to carry out SDS-PAGE and immunoblotting.

DS6 immunoprecipitation goes out the protein band of > 250kDa, can see this protein belt (Fig. 5 A, B and C) in antigen-positive cell lysate.In some clones (i.e. T-47D), observe doublet.In the Caov-3 immunoprecipitate processed with neuraminidase or periodic acid (Fig. 5 A and B), the band of > 250kDa is destroyed, and this shows that CA6 epi-position is positioned on the band of > 250kDa.The band of > 250kDa also demonstrates the N-glycanase process of immunoprecipitate insensitive, and it is consistent (Fig. 5 F) that this and CA6 are positioned at that O-joins on sugar.In addition, support 250kDa band be CA6 antigen be such fact, namely in DS6 antigen negative cells, DS6 does not have immunoprecipitation to go out such band (Fig. 5 D and E).

CA6 antigen is Muc1 to have several evidences to show.Due to its high molecular and susceptibility O-being joined to sugared specific sugar lytic enzyme, CA6 antigen is likely Saliva Orthana.Mucinous overexpression is particularly well characterized in tumour in mammary gland and ovarian tumor, and this is consistent with the primary tumor reactive behavior of DS6.In addition, as CanAg (it is the sialic acid sugar epi-position on Muc1), CA6 is insensitive to perchloric acid precipitation, and this shows that CA6 antigen may be greatly that (heavily) O-is glycosylated.Observe in some DS6 express cell systems, DS6 immunoprecipitation goes out the doublet of > 250kDa, shows that CA6 is Muc1.The feature of mankind Muc1 is the different Muc1 allelotrope of existence two kinds, and number that its series winding repeats is different, and this causes the Muc1 protein expression of two kinds of different molecular weights.

Whether on Muc1, find CA6 to check, SDS-PAGE is carried out to the DS6 immunoprecipitate from Caov-3 lysate, and carry out immunoblotting with DS6 or Muc1 VNTR antibody CM1.As viewed at Fig. 6 A, CM1 with by DS6 immunoprecipitation > 250kDa band generation kickback out.In fig. 6b, when carrying out immunoblotting with DS6 or CM1, DS6 and the CM1 immunoprecipitate from HeLa cell lysate demonstrates the doublet of same > 250kDa.These results show that CA6 epi-position is positioned on Muc-1 protein really.In HeLa (and T-47D) cell, viewed DS6 doublet can by such facts explain, and namely the expression of Muc-1 is instructed by the not isoallele of the tandem repeat sequence with different number.

Although CM1 and DS6 is in conjunction with identical Muc-1 albumen, but they are different epi-positions.The Dot blot of Caov-3 lysate, by the Chemical deglycosylation effect of trifluoromethanesulfonic acid (TFMSA), destroys DS6 signal (Fig. 3).But this identical process enhances CM1 signal.Deglycosylation may be the epi-position exposing the CM1 antibody be hidden.In addition, the flow cytometry of DS6 and CM1 shows in conjunction with the comparison (Fig. 4) of result, CA6 epi-position be not be present in express Muc1 each cell on.Find enjoyably, CA6 epi-position is not expressed (table 3) on Colo205, known Colo205 expression of cell lines high-caliber Muc1 CanAg sialic acid sugar epi-position.

Table 4

* the maximum average relative fluorescence of MMF=

embodiment 4: the quantitative analysis of wandering (shed) CA6 epi-position

Because CA6 epi-position is positioned on Muc1, known in many cancer patientss, Muc1 molecule can scatter in blood flow, so take quantivative approach, object determines whether such level can suppress DS6 Antybody therapy.The combination of circulating antibody and antigen is considered to cause immunocomplex to be removed fast from blood.If the antibody dosage be applied has very most of by removal from circulation rapidly, then the amount arriving tumour probably reduces, and this causes the anti-tumor activity of Antybody therapy agent to reduce.When antibody and efficient cytotoxic compound coupling, the quick removing of conjugate may increase non-specific toxicity potentially.Therefore, for the drug conjugates of antibody-little, such as DS6-DM1, can expect, high-caliber wandering antigen can reduce anti-tumour effect and increase dose limiting toxicity (dose-limiting toxicity).

The clinical trial of recent Antybody therapy has obtained the information about the wandering dynamic (dynamical) impact of Ag concentration against drug.Such as, at Herceptin (Herceptin)---it is be used for the treatment of the antibody of transitivity breast cancer of expressing her2/neu---clinical trial in, when wandering Her2/neu level is at below 500ng/mL, the pharmacokinetics display that Herceptin is removed is not changed (Pegram etc., J.Clin.Oncol.16 (8): 2659-71 (1998).Assuming that the molecular weight of the Her2/neu scattered is 110,000 dalton, the volumetric molar concentration of wandering Her2/neu seems almost not affect pharmacokinetics lower than 4.5nM.

In another example, the clinical trial of cantuzumab mertansine (huC242-DM1) is adopted to show, before treatment, the level of wandering CanAg (C242 epi-position) does not associate (Tolcher etc., J.Clin.Oncol.21 (2): 211-22 (2003) with the pharmacokinetics of cleaning antibody.CanAg epi-position, similar to the CA6 epi-position identified by DS6, the tumour-specific O for the uniqueness on Muc1 joins sialic acid sugar epi-position.But the heterogeneous character of CanAg epi-position makes it be difficult to use mole term quantitative.In general groups, Muc1 allelotrope changes in length, and this depends on the number that the series winding repeated in (VNTR) structural domain at the series winding of variable number repeats.The site of several O-linked glycosylation effect can be there is in each series winding repeats.Iuntercellular change in intrinsic glycosyl transferase activity imparts CanAg and expresses complicacy.Therefore, even at a patient, the CanAg epi-position that each Muc1 molecule has broad range is also possible.And in a group patient, the ratio of the CanAg epi-position of each Muc1 molecule will be different.For this reason, measure the wandering CanAg in serum sample by sandwich ELISA, the wandering Muc1 wherein with CanAg epi-position is caught by C242, and is detected by biotinylation C242/ streptavidin HRP system.The CanAg scattered out by quantitatively with standard unit (U), is directly proportional to the epi-position number of every ml serum, but not is represented by the volumetric molar concentration of Muc1.Similarly, for wandering CA6 epi-position, quantitatively there is similar situation.On the contrary, for Herceptin, each wandering her2/neu molecule only has an epi-position, and what this greatly simplify wandering antigen is quantitative.

In order to be scattered by CA6, epitope levels and those results found in the clinical trial of Herceptin and cantuzumab mertansine connect, and develop the method for the epi-position such as volumetric molar concentration of sialic acid sugar epi-position of scattering for the complexity obtained on Muc1.First, the simple sandwich ELISA assay to DS6 is set up.The description of this analysis is shown in Fig. 7 A.DS6 is used to catch the Muc1 with CA6 epi-position.Because each Muc1 molecule has multiple CA6 epi-position, biotinylated DS6 is also used as tracer antibody.By streptavidin-HRP, use ABTS as substrate, detect the biotinylation DS6 be combined with captured CA6.CA6 epi-position is caught from serum of ovarian cancer patients or the standard substance from commercially available Muc1 test kit (CA15-3), and these standard substance are for monitoring the wandering Muc1 of patients with mastocarcinoma.Arrange arbitrarily DS6 unit/ml, it is equal to CA15-3 standard unit/ml.

Show the result of DS6 sandwich ELISA in figure 7b, which use CA15-3 standard substance.The curve produced is closely similar with the curve obtained with CA15-3 standard substance in CA15-3 analyzes.For DS6 unit/ml being converted into the volumetric molar concentration of CA6, need biotinylation DS6 typical curve signal being converted into pik DS6.Assuming that be man-to-man stoichiometry between CA6 epi-position and biotinylation DS6 antibody, and the molecular weight of biotinylation DS6 is 160,000 dalton, then can calculate the mole number of the CA6 that added every volume sample is caught.

The two kinds of alternative methods producing the typical curve representing biotinylation DS6 are described in figure gA and B.In fig. 8 a, sheep anti-mouse igg polyclonal antibody is used to catch biotinylation DS6, and it is detected in the mode identical with mode used in sandwich ELISA assay shown in the figure 7 again.In the method shown in Fig. 8 B, biotinylation DS6 is directly taped against on elisa plate, and is detected as shown in fig. 8 a.As seen in Fig. 8 C, the biotinylation DS6 typical curve produced by each method is very consistent.

The displayed in Table 5 analysis that the various wandering antigen of serum of ovarian cancer patients's sample is done.The treatment of monitoring ovarian cancer patients is generally used for by measuring CA125 unit/ml, the CA125 ELISA scattered out.The CA125 situation of serum sample is provided.Use the capture antibody and detection antibody that identify and be different from the epi-position that DS6 identifies, be generally used to the treatment of monitoring patients with mastocarcinoma by unit/ml, the CA15-3 ELISA measuring wandering Muc1.In table 5, the CA15-3 in serum of ovarian cancer patients's sample is measured.

Table 5

¹measured by commercial ELISA Kit

²(1 CA15-3 U=1 DS6 U) is measured by business CA15-3 standard substance

³sheep anti-mouse igg & vitamin H-DS6 typical curve

⁴vitamin H-DS6 typical curve

For the value of the CA15-3 reported in table 5, employ the commercially available CA15-3 enzyme immunoassay test kit (Enzyme Immuno Assay kit) from CanAg Diagnostics.For DS6 unit/ml, in DS6 sandwich ELISA, CA15-3 standard substance (the CA15-3 enzyme immunoassay test kit from CanAg Diagnostics) is used to produce typical curve.DS6 unit/ml is set arbitrarily and equals CA15-3 unit/ml.In the end in two row, be used in the biotinylation DS6 typical curve shown in Fig. 8 C to calculate the wandering CA6 of picomole (pM).

For the quantitative analysis of CanAg level, the CanAg serum level reported is for the patient participating in cantuzumab mertansine clinical trial before the treatment (Tolcher etc., J.Clin.Oncol.21 (2): 211-22 (2003).Use CanAg standard substance, use the ELISA test be similar to described in DS6, make CanAg typical curve.C242 is used to catch CanAg standard substance.Use biotinylation C242 tracer, then use ABTS to develop as substrate streptavidin-HRP, complete the detection of captured CanAg.Set up biotinylation C242 typical curve, as to biotinylation DS6 do, with the volumetric molar concentration making unit/ml be converted into circulation CanAg epi-position.In table 6, report the CanAg level from cantuzumab mertansine clinical trial patient, and the corresponding calculating volumetric molar concentration of circulation CanAg.

Table 6

¹level before the process of the circulation CanAg measured by sandwich ELISA

²sheep anti-mouse igg & vitamin H-C242 typical curve

³vitamin H-C242 typical curve

The pM level of the wandering CA6 of ovarian cancer patients with show the comparing of calculating pM level of the wandering CanAg of CanAg positive cancer patients, usually, the CA6 level of scattering out and the CanAg level of scattering out are similar.In addition, only there are 2 examples may have the CA6 level of more than 4.5nM in the serum sample of 16 routine ovarian cancer patients, (serum sample 5 and 9, their signal is beyond the scope of typical curve), on this level, observe in the clinical trial of Her2/neu positive breast cancer patient and can change herceptin pharmacokinetics.In 37 routine clinical trial patients, 3 examples are only had to be found CanAg level higher than 4.5nM.In this clinical trial, wandering CanAg level does not associate with removing sooner of cantuzumab mertansine.But, the patient with the highest CanAg level (31240U/ml) only after infusion 8 hours sampled.These results show, some epi-positions of Muc1, such as CA6 and CanAg, although scatter out in cancer patients, its level of scattering out does not reach the level suppressing Antybody therapy agent treatment.

embodiment 6: clone mouse DS6 antibody variable region

In clinical setting, mouse monoclonal antibody such as DS6 has limited effectiveness, because they are identified as xenobiotics by human immune system.Patient generates human antimouse antibody (human anti-mouse antibodies (HAMA)) very soon, and this causes the quick removing of murine antibody.Therefore, the variable region of mouse DS6 (muDS6) by surface reconstruction, with generating humanized DS6 (huDS6) antibody.

Mouse DS6 antibody variable region is cloned by RT-PCR.Use Qiagen RNeasy Miniprep Kit, be purified into total serum IgE from the T175 bottle that converges of DS6 hybridoma.By UV spectrophotometry RNA concentration, use Gibco Superscript II test kit and random hexamers, carry out RT reaction with 4-5 μ g total serum IgE.

PCR reaction is carried out, based on Wang Z etc., J Immunol Methods.Jan 13 with degenerated primer; Described in 233 (1-2): 167-77 (2000).RT reaction mixture is used directly to degenerate pcr reaction.Use 3 ' light chain primer HindKL,

(TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC)(SEQ IDNO：25)

With 3 ' heavy chain primer BamIgGl,

(GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC)(SEQ ID NO：26)，

For the PCR primer of 5 ' end, the primer of light chain is SaclMK

(GGGAGCTCGAYATTGTGMTSACMCARWCTMCA)(SEQ ID NO：27)，

The primer of heavy chain is

EcoR1MH1 (CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC) (SEQ ID NO:28) and

Equal mixture (mixing base: the H=A+T+C of EcoR1MH2 (CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG) (SEQ ID NO:29), S=G+C, Y=C+T, K=G+T, M=A+C, R=A+G, W=A+T, V=A+C+G, N=A+T+G+C).

PCR reaction is standard, what make an exception is supplement PCR with 10%DMSO to react (50 μ l reaction mixtures, containing, for example under final concentration material: 1 × reaction buffer (ROCHE), each dNTP of 2mM, each primer of 1mM, 2 μ l RT reaction product, 5 μ l DMSO and 0.5 μ l Taq (ROCHE)).Use amendment from Wang Z etc., (J Immunol Methods.Jan 13; 233 (1-2): 167-77 (2000)) program, on MJ research thermal cycler, carry out PCR reaction: 1) 94 DEG C, 3 minutes; 2) 94 DEG C, 15 seconds; 3) 45 DEG C, 1 minute; 4) 72 DEG C, 2 minutes; 5) step 2 is circulated back to, 29 times; 6) terminate 72 DEG C of final extension steps with 10 minutes.By the Restriction Enzyme produced by PCR primer, PCR primer is cloned in pBluescript II SK+ (Stratagene).Seqwright order-checking service is checked order to heavy chain and light chain clone.

In order to confirm 5 ' end cDNA sequence, carry out other PCR and clone.Be placed into the Blast search website of NCBI by the DS6 light chain of degenerate pcr colony assay and heavy chain cDNA sequences, the murine antibody sequence with the signal sequence submitted to is saved.Utilize the conserved sequence in associated dna sequence, design PCR primer by these signal peptides.EcoRI restriction site is added in leader sequence primer (table 7), and these are used in RT-PCR reaction, as mentioned above.

Table 7

Several independent light chain and heavy chain clone are sequenced, to identify and to avoid the possible sequence errors produced by polysaccharase.Light chain and heavy chain RT-PCR are cloned, only obtains single sequence.These sequences for design can increase extend to mouse DS6 light chain in signal sequence and sequence of heavy chain primer be enough.Clone subsequently from these PCR reactions continued confirms 5 ' end sequence of variable region, and it was once changed by initial degenerated primer.The accumulation result of cloning from various cDNA provides final mouse DS6 light chain presented in fig .9 and sequence of heavy chain.Use Kabat and AbM definition, identify three light chains and heavy chain CDRs (Fig. 9 and 10).Show the search of NCBI IgBlast database, muDS6 antibody chain variable region most probable is derived from mouse IgV κ ap4 germline gene, and variable region of heavy chain most probable is derived from mouse IgVh J558.41 germline gene (Figure 11).

the mensuration of the Variable region surface residues of embodiment 7:DS6 antibody

The antibody resurfacing techniques described by (1994) and Roguska etc. (1996) such as Pedersen starts from the surface residue of prediction murine antibody variable sequence.Surface residue is defined as such amino acid, that is, at least 30% of its total surface area can close to water molecules.When lacking analytic structure to find the surface residue of muDS6, we compare (Figure 12) to the antibody that 10 in 127 antibody structure data have most homologous sequence.The solvent accessibility of each Kabat position of these sequences be compared is by average (Figure 13 A and B).

The surface location experience second of average accessibility between 25% to 35% takes turns analysis, is undertaken (Figure 13 A and B) by comparing the antibody subtype containing two identical residue at either side.Second take turns analysis after, it is 23 that 21 of muDS6 heavy chain prediction surface residues are increased, Tyr3 and Lys23 is added in the residue list of the prediction surface accessibility with more than 30%.In our most surfaces reshaped antibody, employ the Kabat definition of heavy chain CDR1, but for DS6, in computation process, employ AbM definition carelessly, therefore heavy chain residues T28 is not defined as skeleton surface residue, otherwise it may be then.The number of light chain surface position reduces to 15 from 16, because take turns in analysis second, the prediction surface accessibility of Ala80 reduces to 27.8% from 30.5%.MuDS6 heavy chain and light chain variable sequence have altogether 38 prediction surfaces can and framework residue.

embodiment 8: people's antibody is selected

Corresponding position in human antibody sequence in the surface location of mouse DS6 variable region and Kabat database is compared (Johnson G, Wu TT.Nucleic Acids Res.Jan1; 29 (1): 205-6 (2001)).Use antibody database management software SR (Searle, 1998) to extract and comparison from native heavy and the right surface residue of light chain people antibody.Select the surface, people antibody variable region with most similar face residue---to being positioned at within position give and special consideration, to replace mouse DS6 antibody variable region surface residue.

embodiment 9: be fitted together to the expression vector with humanized antibody

Light chain and heavy chain paired sequences are cloned in single mammalian expression vector.The PCR primer of people's variable sequence creates restriction site, and this makes people's signal sequence be added in pBluescriptII cloning vector.Then, variable sequence can be cloned in Mammalian expression plasmid, and this plasmid has respectively for EcoRI and BsiWI or HindIII and ApaI site (Figure 14) of light chain or heavy chain.Light chain variable sequence is cloned in people IgKappa constant region with meeting frame, and variable heavy chain sequence is cloned on human IgG ammal constant-region sequences.In final expression plasmid, people's CMV promoter drives the expression of light chain and heavy chain cDNA sequences.

embodiment 10: the qualification of the residue of possible negative impact DS6 activity

Up to the present, in most humanization process, the molecular model of target antibody is established, thus identifies the residue of position closest to CDR as potential problem residue (problem residues).Because the number of studied resurfaced antibody is in continuous increase, therefore historical experience is at least the same effective with modeling in forecasting problem, does not therefore set up the molecular model of DS6.On the contrary, the residue of antibody mouse DS6 surface residue and those previous surface reconstructed compares, have affect antibody binding activity to be low to moderate high risk residue identified.

In obtainable parsing antibody structure and the molecular model from previous humanization work, similar residue group is be positioned at CDR's by repetitive identified within.Use this data, table 1 provide may closest to and may be positioned at CDR's within mouse DS6 residue.In previous humanization, many in these positions are also changed, but only have heavy chain position 74 once to cause binding activities to lose.In huC242 and huB4, mouse residue is retained in this position, and object preserves the binding activities of murine antibody.On the other hand, in humanization 6.2G5C6, this identical position is become corresponding people's residue, and does not lose activity (6.2G5C6 is anti-IGF1-R antibody, is often often simply referred to as anti-C6).Although any residue in table 1 may be all a problem in humanized antibody, but heavy chain residues P73 will receive publicity especially, considers the previous experience about this position.

embodiment 11: the selection on the people surface of most homology

Use SR software, from Kabat antibody sequence database, find out the Candidate human antibody surfaces for surface reconstruction muDS6.This software provides an interface, only search for the specific resi-dues for this antibody database.In order to retain natural pairing, the surface residue of light chain and heavy chain by together with compare.Most homology people surface from Kabat database is arranged by the grade of sequence iden.Front 3 surfaces arranged by SR Kabat database software are provided in table 2.Then these surfaces are compared, to identify that needs are done minimum change to residue identified in Table 1 by who surface.Anti-Rh (D) antibody, 28E4 (Boucher etc., 1997), needs the surface residue of minimal number to change (altogether 11), and only has three in these residues and be included in the list of potential problems residue.Because 28E4 antibody provides the people surface of most homology, it is the best candidate of surface reconstruction muDS6.

embodiment 12: the structure of the DNA sequence dna of humanization DS6 antibody

PCR mutagenesis is used to carry out the change of 11 surface residues of DS6.Clone enterprising performing PCR mutagenesis at mouse DS6 variable region cDNA, build surface reconstruction people DS6 gene.Humanization primer sets is designed to the amino acid change produced needed for surface reconstruction DS6, as shown in Table 8 below.

Table 8

PCR reaction is standard, unlike supplementing described reaction (50 μ l reaction mixtures with 10%DMSO, material containing, for example lower final concentration: 1 × reaction buffer (ROCHE), 2mM often plants dNTP, 1mM often plants primer, 100ng template, 5 μ l DMSO and 0.5 μ l Taq (ROCHE)).They follow these steps to, and MJ Research thermal cycler runs: 1) 94 DEG C, 1 minute; 2) 94 DEG C, 15 seconds; 3) 55 DEG C, 1 minute; 4) 72 DEG C, 1 minute; 5) step 2 is circulated back to 29 times; 6) terminate by 72 DEG C of final extension steps of 4 minutes.Its corresponding Restriction Enzyme digestion of PCR primer, and be cloned in pBluescript cloning vector.Clone is checked order, to confirm that amino acid changes.

Because for a change heavy chain residues P73 caused problem in the past, so establish the heavy chain of two kinds of forms, one has people 28E4 T73, a kind of reservation mouse P73.In the humanization DS6 of two kinds of forms, other 10 surface residues become people 28E4 residue (table 2) from mouse.Be loyal to common naming method, the most humanized is 1.0 forms, because it has all 11 people's surface residues.When other form of needs, the heavy chain form retaining P73 is named as 1.2 forms, and therefore 1.1 forms are left the form comprising maximum mouse residue numbers.The aminoacid sequence of these two kinds of humanization forms of comparing with mouse DS6 aminoacid sequence is shown in Figure 15 A and B.These two kinds of humanization DS6 antibody genes are all cloned into antibody expressing plasmid (Figure 14), for instantaneous and stable transfection.CDNA and the aminoacid sequence of the variable region of light chain of humanization form 1.01 and 1.21 are identical, and are shown in Figure 16.Heavy chain cDNA and the aminoacid sequence of humanization form 1.01 and 1.21 are shown in Figure 17 A and B.

the expression and purification of embodiment 13:huDS6 in Chinese hamster ovary celI and avidity measure

Whether remain the binding affinity of msDS6 to measure humanization DS6 form, it is required for expressing also antibody described in purifying.With the DS6 (chDS6) of chimeric versions thereof with human constant region and mouse variable region, or application huDS6v1.01 or huDS6v1.21 stable transfection Chinese hamster ovary celI system.

By CHODG44 cell (4.32 × 10 ⁶individual cell born of the same parents/plate) be inoculated in Nonsele ctive culture media (α-MEM+ Nucleotide (Gibco), supplement with 4mM L-glutaminate, 50U/ml penicillin, 50 μ g/ml Streptomycin sulphates and 10%v/v FBS) in 15cm plate in, and be positioned over 37 DEG C, 5%CO ₂in moistening incubation case.Second day, the revision of the Polyfect Transfection scheme using Qiagen to recommend, with chDS6 expression plasmid transfectional cell.Nonsele ctive culture media is siphoned away from cell.Plate is washed with 7ml preheating (37 DEG C) PBS, and with 20ml Nonsele ctive culture media plate described in filling again.Plasmid DNA (11 μ g) is diluted in 800 μ lHybridoma SFM (Gibco).Then, 70 μ l Polyfect (Qiagen) are joined in DNA/SFM mixture.Then by this Polyfect mixture vortex a few second gently, and at room temperature incubation 10 minutes.Nonsele ctive culture media (2.7ml) is added in mixture.By the cell culture 24 hours of this final mixture and bed board.

From plate, remove transfection mixture/substratum, then trypsin treatment carried out to cell and count.Then, with different concentration (1800,600,200 and 67 cells/well), selective medium (α-MEM-Nucleotide will be plated on, supplement with 4mM L-glutaminate, 50U/ml penicillin, 50 μ g/ml Streptomycin sulphates, 10%v/v FBS, 1.25mg/ml G418) in cell be placed in 96 orifice plates (250 μ l/ hole).By cell cultures 2-3 week, if necessary, supplemental medium.Quantitative ELISA is used to screen the antibody producing level in each hole.By Immulon2HB 96 orifice plate with goat anti-human igg F (ab) ₂antibody bag is by (Jackson Immunoresearch; 1 μ g/ hole, in 100 μ l 50mM sodium carbonate buffers, pH 9.6), and at room temperature incubation 1.5h, rotate simultaneously.All subsequently step all at room temperature carry out.With T-TBS (0.1%Tween-20, TBS) hole flushing twice, and close 1h with the Block buffer (1%BSA, T-TBS) of 200 μ l.With T-TBS, hole is washed twice.In independently flat board, in Block buffer, by antibody standard substance, EM164 (100ng/ml) and culture supernatant carry out serial dilution (1: 2 or 1: 3).These dilutions (100 μ l) are transferred in elisa plate, and incubation 1h.Hole T-TBS washs three times, and with 100 μ l goat anti-human igg Fc-AP (Jackson lmmunoResearch) incubation 45 minutes, goat anti-human igg Fo-AP was diluted to 1: 3000 with Block buffer.Wash after 5 times with T-TBS, use 100 μ l PNPP developer (development reagent) (10mg/ml PNPP (p-Nitrophenyl phosphate disodium salts; Pierce), 0.1M diethanolamine pH 10.3 damping fluid) is developed the color 25 minutes in hole.The absorbancy at 405nm place is measured in ELISA plate reader.At the absorbance reading (reading for culture supernatants) of the linear portion of typical curve for determining antibody horizontal.

Then subclone, amplification are carried out to the highest production clone that ELISA identifies, and prepare frozen cell liquid storage.

For the expression of huDS6v1.01 and huDS6v1.21, by DG44CHO cell (Dr.LawrenceChasin, Columbia University, NY) (Gibco catalog#12571 in the Alpha MEM with nucleosides and deoxynucleoside is cultivated, Grand Island, New York).Described culture medium supplemented has 10% foetal calf serum (HyClone catalog#SH30071.03, Logan, UT), 1% gentamicin (Mediatech catalog#30-005-CR, Herndon, and 2mM L-glutaminate (L-glut) (BioWhittaker catalog#17-605E VA), Walkersville, MD).This formulation is called CHO perfect medium.

With huDS6 plasmid DNA transfection DG44CHO cell (5x106) of 50 μ g.Before transfection, by cell from trypsin Gibco catalog#15090-046, Grand Island, NY) remove in bottle, with unsupplemented Alpha MEM (the Gibco catalog#12561 lacking nucleosides and deoxynucleoside, Grand Island, NY) wash twice.This is called as washing substratum.Cell and plasmid DNA are mixed in the pole cup (BioRad catalog#1652088, Hercules, PA) at 0.4cm interval.They are placed in 2 minutes on ice, subsequently in BioRad electroporation device with 1,000 μ F and 260 volt acceptance effect.After electroporation, by cell incubated on ice 2 minutes.Subsequently cell is layered on 5 24 orifice plates in CHO perfect medium (Costar catalog#3524), remains on subsequently in 37 DEG C of incubator with 5%CO2.After 48 hours, from hole, remove substratum.With washing substratum washed cell once, and unsupplemented AlphaMEM (the Gibco catalog#12561 added without nucleosides and deoxynucleoside, Grand Island, NY), described MEM is supplemented with foetal calf serum (the Gibco catalog#26400-044 of 1% gentamicin, 2mM L-glut, 10% dialysis, Grand Island, and 1.25mg/mL geneticin (G418) (Gibco catalog#11811 NY), Grand Island, NY).This complete formulation is called Selective agar medium.Selective agar medium incubation will be cloned in about 2 weeks, now by their antibody production of quantitative ELISA screening.Subsequently subclone, amplification are carried out to the clone of production peak, prepare frozen cell storage liquid.

In order to produce the antibody of q.s in order to purifying, by the 30ml selective medium amplifying cells (~ 1 × 10 on 15cm plate being supplemented with Ultra Low IgG FBS (Gibco) ⁶cell/plate), and cultivate 1 week.Culture supernatant collected in 250ml tapered tube, in desk centrifuge, spun down (2000rpm, 5 minutes, 4 DEG C), then carries out sterile filtration by 0.2 μm of filter.

In order to purifying DS6, joined by NaOH in the culture supernatant of filtration, reaching final pH is 8.0.With binding buffer liquid balance Hi Trap rProtein A post (Amersham) of 20-50 column volume.Peristaltic pump is used to be loaded onto on post by supernatant liquor.Then, post is washed with the binding buffer liquid of 50 column volumes.Elution buffer (100mM acetic acid, 50mM NaCl, pH 3) combining antibody is used to be eluted to from post and to be placed in the test tube group of classification collector.With neutralization buffer (2M K ₂hPO ₄, pH 10.0) neutralize the antibody that elutes, and in PBS dialyzed overnight.The antibody of dialysis is filtered by 0.2 μm of syringe filter.Measure the absorbancy at 280nm place, to measure final protein concn.

The avidity of huIgG and muDS6 of purifying is compared by flow cytometry.In battery of tests, measure the direct combination with CA6-express cell system KB.As shown in figure 18, muDS6, chuDS6, huDS6v1.01 and huDS6v1.21 demonstrate closely similar avidity, and its apparent Kd is respectively 0.82nM, 0.69nM, 0.82 and 0.85nM, and this shows that surface reconstruction does not destroy CDRs.Remaining the avidity of muDS6 in order to confirm huDS6 form, having carried out CBA.The advantage of this mode is that same detection system is used to mouse and people's antibody; I.e. vitamin H-muDS6/ streptavidin-DTAF.Relatively the result of the CBA of muDS6, chuDS6, huDS6v1.01 and huDS6v1.21 and vitamin H-DS6 competitive capacity is shown in Figure 19.The apparent EC50 of muDS6, chuDS6, huDS6v1.01 and huDS6v1.21 is respectively 1.9nM, 1.7nM, 3.0 and 1.9nM.These results illustrate, the surface reconstruction to muDS6 in order to generating humanized DS6 does not almost cause binding affinity to reduce.

the preparation of embodiment 14:DS6-DM1 cytotoxic conjugate

N-succinimide-4-(the 2-pyridine dithio) valerate (SPP) of 8 times of molar excess is used to modify muDS6 antibody (8mg/ml), to introduce two thiopyridines bases.At room temperature, in 95%v/v buffer A (50mMKPi, 50mM NaCl, 2mM EDTA, pH 6.5) and 5%v/v DMA, 2h is carried out in reaction.By NAP or Sephadex G25 post (balancing in buffer A), the reaction mixture expanded a little is carried out gel-filtration.By the 2-mercaptopyridine (Spy) of measuring the absorbancy of antibody at 280nm place and DTT release 280 and the absorbancy at 343nm place, the degree of mensuration modification.Then with 2.5mg Ab/mL, using relative to Spy is the N of 1.7 times of molar excess ^{2 '}-deacetylation-N- ^{2 '}-(3-sulfydryl-1-oxygen propyl group)-maytenin (L-DM1) carries out coupling to the muDS6 modified.This reaction is carried out in buffer A (97%v/v) and DMA (3%v/v).At room temperature cultured overnight will be reacted, ~ 20h.Centrifugal opaque reaction mixture (1162 × g, 10 minutes), then supernatant liquor is passed through to carry out gel-filtration with NAP-25 or S300 (Tandem 3,3x 26/10 desalting column, the G25 medium) post that buffer B (1X PBS pH 6.5) balances.Remove precipitation.Use 0.22 μm of Millex-GV filter by conjugate sterile filtration, then dialyse with Slide-A-Lyzer in buffer B.Filtering the rear absorbancy of material at 252nm and 2g0nm place by measuring, measuring the number of the DM1 molecule that per molecule muDS6 connects.DM1/Ab ratio is 4.36, and the stage yield of coupling MUDS6 is 55%.Coupled antibody concentration is 1.32mg/mL.By size exclusion chromatography (SEC), biochemical characterization is carried out to the conjugate of purifying, find that this conjugate 92% is monomer.DM1 in the conjugate of purifying analysis display, 99% with antibody covalent attachment.In fig. 20, the fluidic cell binding analysis of muDS6 and the Caov-3 cell of muDS6-DM1 conjugate and unmodified shows, the coupling of muDS6 causes avidity only to have a little loss.

embodiment 15:muDS6-DM1 cytotoxicity in vitro

As naked antibody, muDS6 has been presented in cell culture and has not bred or growth inhibitory activity (Figure 21).But when when there is DM1 and being coupled to sheep anti-mouse igg heavy chain and light chain, during by cell and muDS6 incubation, this conjugate target is effectively sent on cell by muDS6, causes indirectly cytotoxicity (Figure 21).In order to measure the intrinsic activity of naked muDS6 further, using muDS6 to carry out CDC (CDC) and measuring.When the various dilution of existence 5% people or rabbit anteserum and muDS6, HPAC and ZR-75-1 cell (25000 cells/well) is placed into 96 orifice plates, at 200 μ l RHBP substratum (RPMI-1640,0.1%BSA, 20mM HEPES (pH 7.2-7.4), 100U/ml penicillin and 100ug/ml Streptomycin sulphate) in.At 37 DEG C of Incubate cells 2h.Then Alamar Blue (final concentration of 10%) reagent (Biosource) is added in supernatant liquor.Before measurement fluorescence, by cell culture 5-24 hour.In CDC (CDC) measures, mouse DS6 does not act on (Figure 22).This shows that the treatment use of muDS6 needs in conjunction with toxic effect molecule.

With different DS6 positive cell lines, two kinds of different mensuration forms are used to check the cytotoxicity of the DS6 antibody of CHROMATOGRAPHIC FRACTIONATION AND MASS coupling.Carried out colony formation analysis, wherein cell (1000-2500 cells/well) is laid down in 6 orifice plates, is in the 2ml conjugate with substratum dilution.Under several concentration, generally 3 × 10 ^-11m is to 3 × 10 ^-9between M, cell is continued to be exposed to conjugate, and at 37 DEG C, 6%CO ₂incubation 5-9 days in wet box.Use PBS washing hole, and with 1%w/v Viola crystallina/10%v/v formaldehyde/PBS solution dyeing colony.Then from hole, fully wash away unconjugated dyestuff with distilled water, and make plate dry.LeicaStereoZoom 4 dissecting microscope is used to count colony.

Plant tray efficiency (plating efficiency) (PE) and be calculated as colony number/by the number of bed board cell.Surviving fraction is calculated as the PE of the PE/ untreated cell of process cell.By drawing cell survival fraction to the volumetric molar concentration of conjugate, measure IC ₅₀concentration.In colony formation test (Figure 23), muDS6-DM1 is effective to killing Caov-3 cell, the IC of its estimation ₅₀for 800pM.Concentration is 3 × 10 ^-9the conjugate of M only has impact a little to antigen negative cells A375, and 3 × 10 ^-9m is the maximum concentration of tested muDS6-DM1, and this shows that the cell killing activity of conjugate is specifically for antigen-expressing cells.But although be obviously responsive to maytenin, other DS6 positive cell lines many are not responsive especially to immune conjugate.All cervical cell systems (HeLa, KB and WISH) are all responsive to this conjugate, but, only have and select the ovary of number and cell line of mammary gland to demonstrate any cytotoxic effect.Pancreatic cell system seems that none is affected.

In MTT analyzes, cell is with in implanted 96 orifice plates of the density of 1000-5000 cells/well.Cell is laid down in plate, and naked muDS6 or muDS6-DM1 immune conjugate 200 μ l substratum do serial dilution.The same three parts of operations of sample.Then by cell and antibody/conjugate mixture incubation 2-7d, now analyzed by MTT ([3 (4,5-dimethylthiazole-2-base)-2,5-diphenyltetrazolium bromide]) and evaluate cell survival rate.MTT (50 μ g/ hole) is added in culture supernatant, and makes it at 37 DEG C of incubation 3-4h.Removing substratum, is dissolved in MTT formazan in DMSO (175 μ l/ hole).Measure the absorbancy at 540-545nm place.In MTT cell survival rate is analyzed (Figure 24 C), immune conjugate can effectively kill Caov-3 cell, and it estimates IC ₅₀for 1.61nM.Compared with the naked antibody not having effect, there is the cell (Figure 21 and Figure 24) of Kong Zhongwei containing survival of the conjugate of maximum concentration.

The result that MTT in other clone analyzes different slightly (Figure 24 A, B and D-I).In many cases, although observe some cytotoxicities, conjugate can not kill whole cell mass (except WISH cell) completely.BT-20, OVCAR5 and HPAC cell has resistance especially: in the highest conjugate concentration (32nM) hole, the cell more than 50% is still alive.

embodiment 16: conjugate anti-tumor activity in body

In order to prove muDS6-DM1 conjugate activity in vivo, in SCID mouse, establish people's tumor xenograft.The subcutaneous model of human cervical carcinoma cell system KB is established.KB cell grows in vitro, collect and by 5 × 10 ⁶under individual cell injects the right shoulder of every mouse in 100 μ L serum free mediums, and make it grow 6 days, mean tumour volume reaches 144 ± 125mm ³, now start drug treating.Mouse is given PBS by vein every day, the conjugate of 150 μ g/kg DM1, or the conjugate of 225 μ g/kg DM1 (often organizing two mouse), totally 5 days.Toxic reaction is monitored every day at treatments period.In research process, monitoring gross tumor volume (Figure 25 A) and corresponding body weight (Figure 25 B).

Rapid with the KB tumor growth of PBS control treatment, its doubling time is approximately 4 days.On the contrary, for 225 μ g/kg and 150 μ g/kg dosage groups, 14 days and 18 days respectively after process starts, all demonstrate tumor regression completely with two groups of mouse of conjugate process.Under 150 μ g/kg dosage, tumour postpones about 70 days.Process under 225 μ g/kg causes curing, because when within the 120th day, research stops, without the sign of tumor recurrence.As viewed in Figure 25 B, the mouse display imponderability loss in 150 μ g/kg groups, shows that dosage is able to good tolerance.At higher dosage, only there is the reduction of provisional 3% in the body weight of mouse.At the treatments period of 5 days, mouse did not show obvious signs of toxicity.In sum, this research shows, and muDS6-DM1 process can cure with non-toxic dosage the mouse having KB xenograft tumours.

One group of enterprising pacing examination DS6-DM1 activity (see Figure 26) of subcutaneous xenograft model.The tumor cell line being used to prepare heterograft shows external maytenin susceptibility and the CA6 epitope density (table 9 below) of certain limit.OVCAR5 cell and TOV-21G are ovarian tumor cell system; HPAC is pancreatic tumor cell system; HeLa is tumor of cervix clone.OVCAR5 and TOV-21G cell has low surface C A6 expresses; The surface C A6 that HeLa cell has medium level expresses; HPAC cell has the CA6 density of high surface expression.TOV-21G and HPAC cell is responsive to maytenin; OVCAR5 and HeLa cell to the few 2-7 of the susceptibility of maytenin doubly.

Table 9

^*average maximum relative mean fluorescent

Four kinds of clones are cultivated in vitro, collect, by 1 × 10 in 100 μ L serum free mediums ⁷under individual cell injects the right shoulder of every mouse (each model 6 mouse), and make it grow 6 days, for test group and the control group of OVCAR5, mean tumour volume reaches 57.6 ± 6.7 and 90.2 ± 13.4mm respectively ³, for test group and the control group of HPAC, reach 147.1 ± 29.6 and 176.2 ± 18.9mm respectively ³, for test group and the control group of HeLa, reach 194.3 ± 37.2 and 201.7 ± 71.7mm respectively ³, for test group and the control group of TOV-21G, reach 96.6 ± 22.8 and 155.6 ± 13.4mm respectively ³, now start pharmacological agent.For each model, with PBS vein treatments three the contrast mouse of twice weekly dose, with conjugate (600 μ g/kgDM1) vein treatments three the test mouse of twice weekly dose.Monitor toxic reaction every day at treatments period, in research process, monitor gross tumor volume and body weight.Conjugate effect for various model is shown in Figure 26 A, C, E and G by figure, and corresponding body weight is plotted in Figure 26 B, D, F and H.OVCAR5, TOV-21G and HPAC clone forms invasive tumour, observed by contrasting as the PBS at each model.Before start index growth, HeLa model has the lag-phase of about 3 weeks.In all models, the process of DS6-DM1 conjugate causes occurring tumor regression completely in all mouse.For TOV-21G, HPAC and HeLa model, mouse kept without tumour at the 61st day.In OVCAR5 model, after tumor inoculation about 45 days, tumor recurrence.Therefore, muDS6-DM1 process in the model causes tumor growth delay about 34 days.Delayed growth is significant, because OVCAR5 cell is more insensitive to maytenin, and has low CA6 epi-position expression.In some models, the higher or model of CA6 epitope density has larger maytenin susceptibility, and tumor regression is then more powerful.It should be noted that only application of two doses, this is very important.Clearly, in this research, the dosage of application does not have toxicity to mouse, because do not observe weight loss.Then may realize curing with extra or higher conjugate dosage.

Human ovarian cancer is the disease of peritonaeum mostly.OVCAR5 cell is grown to aggressive intraperitoneal (IP) model in SCID mouse, forms tumor nodule, and produces ascites in the mode similar to human diseases.In order to prove the activity in IP model, muDS6-DM1 is used to treat the mouse (Figure 27) with OVCAR5 IP tumour.OVCAR5 cell grows in vitro, results, and by 1 × 10 in 100 μ L serum free mediums ⁷individual cell carries out peritoneal injection.Make tumor growth 6 days, now begin treatment.Or process mouse weekly, totally two weeks with PBS or the DS6-DM1 conjugate that is used in 600 μ g/kg DM1 dosage, and monitor the body weight loss caused by disease of peritoneum.During by 28 days, the mouse of PBS group have lost the body weight of more than 20%, and it is condemned to death.At the 45th day, put to death after the body weight loss for the treatment of group is more than 20%.This research shows, muDS6-DM1 can postpone the tumor growth in aggressive OVCAR5 IP model, although there is such fact, that is, OVCAR5 cell is more insensitive to maytenin, and each cell has little CA6 epi-position.Because dosage used does not cause visible toxicity sign, so extra or higher dosage may be used to realize further tumor growth delay or healing.

the Synthesis and characterization of embodiment 17:DS6-SPP-MM1-202 Taxoid cytotoxic conjugate

MuDS6 is modified with N-sulfosuccinimide base 4-nitro-2-pyridine-valerate (SSNPP) joint.The SSNPP in the dma of 10 equivalents is joined in the 50mgmuDS6Ab in 90% buffer A, 10%DMA.The final concentration of Ab is 8mg/ml.At room temperature, by reaction stirring 4 hours, then by G25 chromatography purification.Be used in the absorbancy at 280nm (antibody) and 325nm (joint) place, the degree of metric measurement antibody modification, find that there are 3.82 joint/antibody.Antibody is recovered as 43.3mg, and yield is 87%.Coupling muDS6-nitroSPP conjugate is carried out with Taxoid MM1-202 (1812 P.16).This coupling is carried out with the scale of 42mg in 90% buffer A, 10%DM1.During about 20 hours, taxoid is added with the 0.43eq/ joint of 4 equal portions (every equal portions).Now, reaction has become obvious muddiness.After G25 purifying, the conjugate obtained, it is recovered with the yield of about 64%, has about 4.3 taxoid/Ab, the unreacted joint residue of about 1 equivalent.In order to stop unreacted joint, the halfcystine of 1 equivalent/unreacted joint is added in conjugate, simultaneously stirred overnight.Add fashionable at halfcystine, certain yellow tone is significant, this demonstrates the release of thiopyridine.Then hemodialysis reaction solution in buffer B/0.01%Tween 20, then dialyses a couple of days further in independent buffer B.Final conjugate has 2.86 medicine/antibody.Antibody yield is 14.7mg, altogether produces the yield of 35%.Carry out biochemical characterization to conjugate further by SEC, it is found to have the aggregate of 89% monomer, 10.5% dipolymer and 0.5% more high molecular.

Flow cytometry compares muDS6-SPP-MM1-202 taxoid and the combination of muDS6 antibody on HeLa cell, and result is shown in Figure 28.This result shows, and when muDS6 is coupled to Taxan, it remains binding activities.

embodiment 18: the in vitro and in vivo of humanization DS6 conjugate is active

Build huDS6v1.01-SPDB-DM4 conjugate.This conjugate is similar to the muDS6-SPP-DM1 conjugate described in embodiment 14, except the structure of the joint in conjugate/maytenin drug moiety around disulfide linkage is different; MuDS6-SPP-DM1 conjugate has a methyl steric hindrance on two sulphur carbon of the antibody side of joint, and SPDB-DM4 conjugate has two methyl steric hindrances on two sulphur carbon of the maytenin side of joint.

HuDS6v1.01 antibody (8mg/ml) N-succinimido-4-(the 2-pyridyl dithio) butyric ester (SPDB) that 8 times of mole numbers are excessive is modified, to introduce dithiopyridines base.Reaction is carried out 1.5 hours under room temperature in 95%v/v buffer A (50mMKPi, 50mM NaCl, 2mM EDTA, pH 6.5) and 5%v/v ethanol.Reaction mixture is by 15ml Sephadex G25 post (balancing in buffer A) gel-filtration.By measuring the 2-mercaptopyridine (Spy) of antibody in the absorbancy of 280nm and DTT release 280 and the absorbancy of 343nm, measure degree of modification.Then with 1.8mg Ab/mL, using relative to Spy is the N of 1.7 times of molar excess ^{2 '}-deacetylation-N- ^{2 '}-(4-methyl 4-sulfydryl-1-oxygen phenyl)-maytenin (L-DM4) carries out coupling to the DS6 modified.This reaction is carried out in buffer A (97%v/v) and DMA (3%v/v).Reaction is incubated overnight at room temperature, ~ 20h.Relative to muDS6 coupling, this reaction mixture is limpid, and the NAP15ml 25G post immediately by balancing at citrate buffer (20mM Citrate trianion, 135mM NaCl, pH 5.5) carries out gel-filtration.Use 0.22 μm of Millex-GV filter by conjugate sterile filtration.By measuring the absorbancy of material at 252nm and 280nm place of filtering, measure the number of the DM4 molecule that per molecule DS6 connects.Find that DM4/Ab ratio is 3.2, the stage yield of coupling DS6 is 69%.The antibody concentration of coupling is 1.51mg/mL.By size exclusion chromatography (SEC), biochemical characterization is carried out to the conjugate of purifying, find that this conjugate is 92.5% monomer.The analysis display of the DM4 in the conjugate of purifying, > 99% and antibody covalent attachment.

In Figure 29 A, the fluidic cell of DS6 and the KB cell of huDS6v1.01-DM4 conjugate and unmodified combines and shows, the coupling of huDS6v1.01 makes avidity substantially not lose.Substantially described combination is carried out as shown in figure 20, except application KB cell instead of CaOv-3 cell.Substantially as described in Figure 24 G, measure the in vitro toxicity of huDS6v1.01.HuDS6v1.01 kills WISH cell, IC ₅₀4.4 × 10 ^-10m, and non-coupling huDS6v1.01 does not show cellular cytoxicity activity.

The activity in vivo of huDS6v1.01-DM4 measures in HPAC pancreas model.At the 0th day inoculation HPAC cell, carried out immunoconjugates process at the 13rd day.Once gross tumor volume is more than 1000mm ³, put to death PBS control animal.Give conjugate with the dosage of 200 μ g/kg or 600 μ g/kg DM4, this corresponds respectively to antibody concentration 15mg/kg and 45mg/kg.Gross tumor volume (Figure 30 A) and the body weight (Figure 30 B) of mouse is monitored during studying.HuDS6v1.01-DM4 shows effective antitumour activity when 200 μ g/kg DM4, and all mouse obtain tumor regression completely.Identify that the contrast B4-DM4 conjugate of the antigen that HPAC xenograft is not expressed is in the essentially no activity of 200 μ g/kg.Mouse Weight end is lost (Figure 30 B) and is shown, with 200 μ g/kg conjugate process lower than maximum tolerated dose.This result shows, the DS6 of humanization form can mediate the targeted of CHROMATOGRAPHIC FRACTIONATION AND MASS medicine, realizes effective antitumour activity.

The present invention is the following particularly:

1. antibody or its epitope binding fragments, comprise at least one variable region of heavy chain or its fragment and at least one variable region of light chain or its fragment, wherein said variable region of heavy chain or its fragment have the sequence iden of at least 90% with the aminoacid sequence being selected from SEQ ID NO:9, SEQ IDNO:10 and SEQ IDNO:11:

2. the antibody described in the 1st or its epitope binding fragments, the aminoacid sequence of wherein said variable region of heavy chain or its fragment and SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11 has the sequence iden of at least 95%.

3. the antibody described in the 1st or its epitope binding fragments, wherein said variable region of heavy chain or its fragment have the aminoacid sequence of SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

4. the antibody described in the 1st or its epitope binding fragments, wherein said variable region of light chain or its fragment and SEQ ID NO:7 or the aminoacid sequence shown in SEQ ID NO:8 have the sequence iden of at least 90%:

5. the antibody described in the 1st or its epitope binding fragments, the aminoacid sequence of wherein said variable region of light chain or its fragment and SEQ ID NO:7 or SEQ ID NO:8 has the sequence iden of at least 95%.

6. the antibody described in the 1st or its epitope binding fragments, wherein said variable region of light chain or its fragment have the aminoacid sequence of SEQ ID NO:7 or SEQ ID NO:8.

7. the antibody described in the 1st or its epitope binding fragments, wherein said variable region of heavy chain or its fragment have the aminoacid sequence of SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, and wherein said variable region of light chain or its fragment have the aminoacid sequence of SEQ ID NO:7 or SEQ ID NO:8.

8. polynucleotide, its antibody of coding according to the 1st or its epitope binding fragments.

9. polynucleotide, its antibody of coding according to the 4th or its epitope binding fragments.

10. polynucleotide, its antibody of coding according to the 7th or its epitope binding fragments.

11. polynucleotide, the antibody of its coding according to the 1st or the heavy chain of its epitope binding fragments.

12. expression vectors, it comprises the polynucleotide described in the 8th.

13. expression vectors, it comprises the polynucleotide described in the 9th.

14. expression vectors, it comprises the polynucleotide of the 10th.

15. host cells, it comprises the expression vector described in the 12nd.

16. host cells, it comprises the expression vector described in the 13rd.

17. host cells, it comprises the expression vector described in the 14th.

The method of 18. Dispersal risks or its epitope binding fragments, the host cell described in the 15th is cultivated under being included in the condition promoting that described antibody or its epitope binding fragments are expressed, described polypeptide is reclaimed with from described cell culture, wherein said antibody or its epitope binding fragments comprise at least one variable region of heavy chain or its fragment and at least one variable region of light chain or its fragment, and wherein said variable region of heavy chain or its fragment have the sequence iden of at least 90% with the aminoacid sequence being selected from SEQ ID NO:9, SEQID NO:10 and SEQID NO:11:

The method of 19. Dispersal risks or its epitope binding fragments, the host cell described in the 16th is cultivated under being included in the condition promoting that described antibody or its epitope binding fragments are expressed, reclaim described polypeptide with from described cell culture, wherein said variable region of heavy chain or its fragment have the sequence iden of at least 90% with the aminoacid sequence being selected from SEQ ID NO:9, SEQ ID NO:10 and SEQ IDNO:11:

QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLADTSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYWGQGTLVTVSA(SEQ ID NO：10)

QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYWGQGTLVTVSA(SEQ ID NO：11)，

And wherein said variable region of light chain or its fragment and SEQ ID NO:7 or the aminoacid sequence shown in SEQ IDNO:8 have the sequence iden of at least 90%:

The method of 20. Dispersal risks or its epitope binding fragments, the host cell described in the 17th is cultivated under being included in the condition promoting that described antibody or its epitope binding fragments are expressed, described polypeptide is reclaimed with from described cell culture, wherein said variable region of heavy chain or its fragment have the aminoacid sequence of SEQID NO:9, SEQ ID NO:10 or SEQ IDNO:11, and wherein said variable region of light chain or its fragment have the aminoacid sequence of SEQ ID NO:7 or SEQ ID NO:8.

Claims

1. antibody or its epitope binding fragments, comprise at least one variable region of heavy chain or its fragment and at least one variable region of light chain or its fragment, wherein said variable region of heavy chain comprises SEQ IDNO:10 or the aminoacid sequence shown in SEQ ID NO:11.

2. antibody according to claim 1 or its epitope binding fragments, wherein said variable region of light chain comprises the aminoacid sequence shown in SEQ ID NO:8.

3. a cytotoxic conjugate, comprises antibody described in claim 1 or 2 or its epitope binding fragments and cytotoxic agent.

4. cytotoxic conjugate according to claim 3, wherein said cytotoxic agent is CHROMATOGRAPHIC FRACTIONATION AND MASS.

5. cytotoxic conjugate according to claim 3, wherein said CHROMATOGRAPHIC FRACTIONATION AND MASS and described antibody or its epitope binding fragments covalently bound by sour labile bond, photo-labile key, disulfide linkage, peptase labile bond or esterase labile bond.

6. comprise antibody or its epitope binding fragments and the cytotoxic conjugate by the covalently bound cytotoxic agent of SPDB joint, wherein

Described antibody or its epitope binding fragments comprise at least one variable region of heavy chain or its fragment and at least one variable region of light chain or its fragment, and wherein said variable region of heavy chain comprises the aminoacid sequence shown in SEQ IDNO:9 and described variable region of light chain comprises the aminoacid sequence shown in SEQ ID NO:7.

7. cytotoxic conjugate according to claim 6, wherein said cytotoxic agent is Taxan.

8. a monitoring accepts the method for the treatment of the patient with breast cancer of humanization DS6 antibody or its epitope binding fragments, described antibody or its epitope binding fragments comprise SEQ ID NO:1,2,3,4, the CDR sequence shown in 5 and 6, described method comprise make the epi-position of sample and its identification obtained from described patient be different from DS6 identify the anti-CA6 antibody contacts of epi-position.

9. measure a method for the anti-tumor activity of anti-muc1 antibody or its Fab, be included in the external BT-20 of making, BT-483, Caov-3, Caov-4, HeLa, HPAC, HPAF-II, Hs766T, KB, OVCAR5, T-47D, TOV-21G, WISH and ZR75-1 cell and contact with described antibody or its Fab.